FP-Pocket-Binding Effectors and Methods for Using the Same to Modulate Telomerase Activity

ABSTRACT

The present invention embraces compounds selected for interacting with the Fingers-Palm pocket of telomerase and use thereof for modulating the activity of telomerase and preventing or treating diseases or conditions associated with telomerase.

INTRODUCTION

This application is a continuation-in-part of PCT/US2008/080604, filedOct. 21, 2008, which claims benefit of priority to U.S. ProvisionalPatent Application Ser. No. 61/090,726, filed Aug. 21, 2008, and Ser.No. 60/981,548, filed Oct. 22, 2007, the contents of which areincorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

Any organism with linear chromosomes faces a substantial obstacle inmaintaining the terminal sequence of its DNA often referred to as the“end replication problem” (Blackburn (1984) Annu. Rev. Biochem.53:163-194; Cavalier-Smith (1974) Nature 250:467-470; Cech & Lingner(1997) Ciba Found. Symp. 211:20-34; Lingner, et al. (1995) Science269:1533-1534; Lundblad (1997) Nat. Med. 3:1198-1199; Ohki, et al.(2001) Mol. Cell. Biol. 21:5753-5766). Eukaryotic cells overcome thisproblem through the use of a specialized DNA polymerase, calledtelomerase. Telomerase adds tandem, G-rich, DNA repeats (telomeres) tothe 3′-end of linear chromosomes that serve to protect chromosomes fromloss of genetic information, chromosome end-to-end fusion, genomicinstability and senescence (Autexier & Lue (2006)Annu. Rev. Biochem.75:493-517; Blackburn & Gall (1978) J. Mol. Biol. 120:33-53;Chatziantoniou (2001) Pathol. Oncol. Res. 7:161-170; Collins (1996)Curr. Opin. Cell Biol. 8:374-380; Dong, et al. (2005) Crit. Rev. Oncol.Hematol. 54:85-93).

The core telomerase holoenzyme is an RNA-dependent DNA polymerase (TERT)paired with an RNA molecule (TER) that serves as a template for theaddition of telomeric sequences (Blackburn (2000) Nat. Struct. Biol.7:847-850; Lamond (1989) Trends Biochem. Sci. 14:202-204; Miller &Collins (2002) Proc. Natl. Acad. Sci. USA 99:6585-6590; Miller, et al.(2000) EMBO J. 19:4412-4422; Shippen-Lentz & Blackburn (1990) Science247:546-552). TERT is composed of four functional domains one of whichshares similarities with the HIV reverse transcriptase (RT) in that itcontains key signature motifs that are hallmarks of this family ofproteins (Autexier & Lue (2006) supra; Bryan, et al. (1998) Proc. Natl.Acad. Sci. USA 95:8479-8484; Lee, et al. (2003) J. Biol. Chem.278:52531-52536; Peng, et al. (2001) Mol. Cell. 7:1201-1211). The RTdomain, which contains the active site of telomerase is thought to beinvolved in loose associations with the RNA template (Collins & Gandhi(1998) Proc. Natl. Acad. Sci. USA 95:8485-8490; Jacobs, et al. (2005)Protein Sci. 14:2051-2058). TERT however is unique, when compared toother reverse transcriptases in that it contains two domains N-terminalto the RT domain that are essential for function. These include the farN-terminal domain (TEN), which is the least conserved among phylogeneticgroups, but is required for appropriate human, yeast and ciliatedprotozoa telomerase activity in vitro and telomere maintenance in vivo(Friedman & Cech (1999) Genes Dev. 13:2863-2874; Friedman, et al. (2003)Mol. Biol. Cell 14:1-13). The TEN domain has both DNA- and RNA-bindingproperties. DNA-binding facilitates loading of telomerase to thechromosomes while RNA-binding is non-specific and the role of thisinteraction is unclear (Hammond, et al. (1997) Mol. Cell. Biol.17:296-308; Jacobs, et al. (2006) Nat. Struct. Mol. Biol. 13:218-225;Wyatt, et al. (2007) Mol. Cell. Biol. 27:3226-3240). A third domain, thetelomerase RNA binding domain (TRBD), is located between the TEN and RTdomains, and unlike the TEN-domain is highly conserved amongphylogenetic groups and is essential for telomerase function both invitro and in vivo (Lai, et al. (2001) Mol. Cell. Biol. 21:990-1000). TheTRBD contains key signature motifs (CP- and T-motifs) implicated in RNArecognition and binding and makes extensive contacts with stem I and theTBE of TER, both of which are located upstream of the template (Bryan,et al. (2000) Mol. Cell. 6:493-499; Cunningham & Collins (2005) Mol.Cell. Biol. 25:4442-4454; Lai, et al. (2002) Genes Dev. 16:415-420; Lai,et al. (2001) supra; Miller, et al. (2000) supra; O′Connor, et al.(2005) J. Biol. Chem. 280:17533-17539). The TRBD-TER interaction isrequired for the proper assembly and enzymatic activity of theholoenzyme both in vitro and in vivo, and is thought to play animportant role (although indirect) in the faithful addition of multiple,identical telomeric repeats at the ends of chromosomes (Lai, et al.(2002) supra; Lai, et al. (2003) Mol. Cell. 11:1673-1683; Lai, et al.(2001) supra).

Unlike TERT, TER varies considerably in size between species. Forexample, in Tetrahymena thermophila TER is only 159 nucleotides long(Greider & Blackburn (1989) Nature 337:331-337), while yeast harbors anunusually long TER of 1167 nucleotides (Zappulla & Cech (2004) Proc.Natl. Acad. Sci. USA 101:0024-10029). Despite the large differences insize and structure, the core structural elements of TER are conservedamong phylogenetic groups, suggesting a common mechanism of telomerereplication among organisms (Chen, et al. (2000) Cell 100:503-514; Chen& Greider (2003) Genes Dev. 17:2747-2752; Chen & Greider (2004) TrendsBiochem. Sci. 29:183-192; Ly, et al. (2003) Mol. Cell. Biol.23:6849-6856; Theimer & Feigon (2006) Curr. Opin. Struct. Biol.16:307-318). These include the template, which associates loosely withthe RT domain, and provides the code for telomere synthesis, and theTBE, which partly regulates telomerase's repeat addition processivity.In Tetrahymena thermophila, the TBE is formed by stem II and theflanking single stranded regions, and is located upstream and in closeproximity to the template (Lai, et al. (2002) supra; Lai, et al. (2003)supra; Licht & Collins (1999) Genes Dev. 13:1116-1125). Low-affinityTERT-binding sites are also found in helix IV and the templaterecognition element (TRE) of Tetrahymena thermophila TER.

TERT function is regulated by a number of proteins, some of which act bydirect association with the TERT/TER complex, while others act byregulating access of telomerase to the chromosome end through theirassociation with the telomeric DNA (Aisner, et al. (2002) Curr. Opin.Genet. Dev. 12:80-85; Cong, et al. (2002) Microbiol. Mol. Biol. Rev.66:407-425; Dong, et al. (2005) supra; Loayza & de Lange (2004) Cell117:279-280; Smogorzewska & de Lange (2004) Annu. Rev. Biochem.73:177-208; Smogorzewska, et al. (2000) Mol. Cell. Biol. 20:1659-1668;Witkin & Collins (2004) Genes Dev. 18:1107-1118; Witkin, et al. (2007)Mol. Cell. Biol. 27:2074-2083). For example, p65 in the ciliatedprotozoan Tetrahymena thermophila or its homologue p43 in Euplotesaediculatus, are integral components of the telomerase holoenzyme(Aigner & Cech (2004) RNA 10:1108-1118; Aigner, et al. (2003)Biochemistry 42:5736-5747; O′Connor & Collins (2006) Mol. Cell. Biol.26:2029-2036; Prathapam, et al. (2005) Nat. Struct. Mol. Biol.12:252-257; Witkin & Collins (2004) supra; Witkin, et al. (2007) supra).Both p65 and p43 are thought to bind and fold TER, a process requiredfor the proper assembly and full activity of the holoenzyme. In yeast,recruitment and subsequent up regulation of telomerase activity requiresthe telomerase-associated protein Est1 (Evans & Lundblad (2002) Genetics162:1101-1115; Hughes, et al. (1997) Ciba Found. Symp. 211:41-52;Lundblad (2003) Curr. Biol. 13:R439-441; Lundblad & Blackburn (1990)Cell 60:529-530; Reichenbach, et al. (2003) Curr. Biol. 13:568-574;Snow, et al. (2003) Curr. Biol. 13:698-704). Est1 binds the RNAcomponent of telomerase, an interaction that facilitates recruitment ofthe holoenzyme to the eukaryotic chromosome ends via its interactionwith the telomere binding protein Cdc13 (Chandra, et al. (2001) GenesDev. 15:404-414; Evans & Lundblad (1999) Science 286:117-120; Lustig(2001) Nat. Struct. Biol. 8:297-299; Pennock, et al. (2001) Cell104:387-396).

How telomerase and associated regulatory factors physically interact andfunction with each other to maintain appropriate telomere length isunder investigation. Structural and biochemical characterization ofthese factors, both in isolation and complexed with one another, can beused to determine how the interaction of the TRBD domain with stem I andthe TBE of TER facilitate the proper assembly and promote the repeataddition processivity of the holenzyme.

While in vitro and in vivo screening assays have been developed toidentify agents which modulate telomerase activity or telomere binding,focus has not been placed on identifying agents with a degree ofspecificity for particular domains or substrate pockets. See, U.S. Pat.Nos. 7,067,283; 6,906,237; 6,787,133; 6,623,930; 6,517,834; 6,368,789;6,358,687; 6,342,358; 5,856,096; 5,804,380; and 5,645,986 and US2006/0040307.

SUMMARY OF THE INVENTION

The present invention features a compound selected for interacting withthe fingers-palm (FP) pocket of telomerase. In one embodiment, thecompound has a structure of Formula I or Formula II:

In another embodiment, the compound is selected from the group ofcompounds listed in Table 1. Pharmaceutical compositions and methods forusing such compounds to inhibit or stimulate telomerase activity, and inthe prevention or treatment of a disease or disorder associated withtelomerase are also provided. Diseases or disorders embraced by theinvention include, cancer, Alzheimer's disease, Parkinson's disease,Huntington's disease, stroke, osteoporosis, diabetes and age-relatedmacular degeneration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of telomerase (TERT).

FIG. 1A shows the primary of human, yeast and Tetrahymena thermophilaTERT showing the functional domains and conserved motifs. FIG. 1B is theprimary structure and conserved motifs of the Tribolium castaneum TERT.

FIGS. 2A-2C show the sequence alignment and surface conservation ofTribolium castaneum TERT (TRICA; SEQ ID NO:1) compared with TERTs fromvarious phylogenetic groups including mammals such as mouse (SEQ IDNO:2) and human (SEQ ID NO:3); plants such as Arabidopsis thaliana(ARATH; SEQ ID NO:4); fungi such as Saccharomyces cerevisiae (YEAST; SEQID NO:5) and Schizosaccharomyces pombe (SCHPO; SEQ ID NO:6); andprotozoa such as Tetrahymena thermophila (TETTH; SEQ ID NO:7) andEuplotes aediculatus (EUPAE; SEQ ID NO:8) produced by ClustalW2 (Larkinet al. (2007) Bioinformatics 23:2947-2948). Conserved residues in keysignature motifs are indicated. K210 of helix α10 and polar residues(K406, K416, K418, N423) of the “thumb” domain implicated in directcontacts with the backbone of the DNA substrate are also shown.

FIG. 3 illustrates the mode of inhibition of telomerase by TERTmodulators. The two nucleotides bound at the active site of telomeraseare shown. The small molecule inhibitor of telomerase clashing with oneof the nucleotides is also shown.

FIG. 4 depicts TERT RNA template associations. The nucleotide located atthe 5′-end of the RNA template (rC1) is coordinated by Ile196 and Val197of motif 2 and Gly309 of motif B′. rU2 interacts with Pro311 of motif B′and rG3 coordinates the backbone of helix α15 via a water molecule(Wat18).

FIG. 5 depicts TERT telomeric DNA associations. Interactions between thethumb loop and the DNA are mostly backbone and solvent mediated. Also,the side chains of Lys416 and Asn423 that form part of the thumb loopextend toward the center of the ring and coordinate the DNA backbone.

FIG. 6 depicts DNA interactions with the primer grip region and theactive site. Shown is a stereo view of the DNA interactions with motif Eand the active site residues. The tip of the primer grip region (loopshown on left), formed by the backbone of residues Cys390 and Gly391abuts the ribose group of C22 and this interaction guides the 3′-end ofthe DNA at the active site of the enzyme for nucleotide addition. Theactive site bound magnesium ion (sphere) coordinates the DNA backboneformed by the last two nucleotides. The nucleotide binding pocket ofTERT, which is partially occupied by the last DNA nucleotide, is in partformed by the highly conserved residue Val342 and the invariant Tyr256and Gly308.

DETAILED DESCRIPTION OF THE INVENTION

Telomerase, a ribonucleoprotein complex, replicates the linear ends ofeukaryotic chromosomes, thus taking care of the “end of replicationproblem”. Telomerase is composed of three highly conserved domains,TRBD, the reverse transcriptase (RT) domain, and the C-terminalextension thought to represent the putative “thumb” domain of TERT(FIGS. 1A and 1B), which are organized into a ring-like structure (FIG.1C) that shares common features with retroviral reverse transcriptases,viral RNA polymerases and B-family DNA polymerases. Domain organizationplaces motifs implicated in substrate binding and catalysis in theinterior of the ring, which can accommodate seven-to-eight bases ofdouble stranded nucleic acid. Modeling of an RNA/DNA heteroduplex in theinterior of this ring reveals a perfect fit between the protein and thenucleic acid substrate and positions the 3′-end of the DNA primer at theactive site of the enzyme providing evidence for the formation of anactive telomerase elongation complex.

Indeed, as the high-resolution structure of the telomerase catalyticsubunit, TERT, in complex with its RNA-templating region, part of thecomplementary DNA sequence, nucleotides and magnesium ions shows, theRNA-DNA hybrid adopts an A-form helical structure and is docked in theinterior cavity of the TERT ring. Protein-nucleic acid associationsinduce global TERT conformational changes that lead to a decrease in thediameter of the interior cavity of the ring facilitating the formationof a tight telomerase elongation complex. The enzyme, which uses atwo-metal binding mechanism for nucleotide association and catalysis,contains two nucleotides at its active site. One of the nucleotides isin position for attack by the 3′-hydroxyl of the incoming DNA primerwhile the second site appears to hold the nucleotide transiently,allowing for RNA template-dependent selectivity and telomeraseprocessivity.

X-ray crystal structure indicates that the organization of the fingersand palm domains between the substrate-free and substrate-bound TERTmolecules is highly similar, indicating that like the Hepatitis C viralRNA polymerase (NS5B), telomerase has a preformed active site. Thisobservation is surprising since in most polymerases, including the HIVreverse transcriptase, the fingers domain undergoes significantconformational changes referred to as the open and closed states. In HIVreverse transcriptase, conformational rearrangements of the fingersdomain with respect to the palm domain are essential for nucleotidebinding and positioning at the active site of the enzyme and theirabsence from telomerase suggests possible mechanistic differences innucleotide binding and selectivity between these families of enzymes.

The organization of the fingers and palm domains of TERT creates a deep,well-defined, narrow, mostly hydrophobic Fingers-Palm pocket(FP-pocket). The FP-pocket, generated by residues Y170, F193, A195,D254, W302, H304, L307, Q308 (with reference to Tribolium castaneumtelomerase; FIG. 2), opens into the interior cavity of the TERT ring andis solvent accessible. Moreover, the entry of this pocket is located inclose proximity of the active site (D251, D343 and D344 of T. castaneumtelomerase) of the enzyme, which is absolutely essential for telomerasefunction; alanine mutants of any of the three invariant aspartatescompletely abolishes telomerase activity. In so far as the FP-pocket isa stable, well-defined, solvent accessible cavity and is located inclose proximity of the active site of the enzyme, this pocket wasselected as a target for the identification of effector molecules thatmodulate telomerase activity.

Accordingly, based upon the structure of the FP-pocket, an in silicoscreen was carried out to identify small molecules that associate orinteract with this pocket (FIG. 3). Inhibitors of telomerase wereselected for their ability to occlude the active site of telomerase thusinterfering with nucleotide association and therefore telomerereplication. On the other hand, activators were selected for being inclose proximity to the active site of the enzyme without occluding theactive site. Activators could therefore be involved in favorableinteractions with the incoming nucleotide substrate producing a tightand stable catalytic active complex thus increasing telomerase activity.Structurally similar groups of compounds, identified via in silicoscreening as making extensive contacts with the FP-pocket, are listed inTable 1.

TABLE 1 Group Compound 12-(acetylamino)-N-(aminocarbonyl)-3-thiophenecarboxamideN-(3-{[(aminocarbonyl)amino]carbonyl}-2- thienyl)isonicotinamideN-(aminocarbonyl)-2-{[(5-methyl-3-thienyl)carbonyl]amino}-3-thiophenecarboxamideN-(3-{[(aminocarbonyl)amino]carbonyl}-2-thienyl)-2-oxo-2H-chromene-3-carboxamide ethyl2-[(4-amino-4-oxo-2-butenoyl)amino]-4,5-dimethyl-3- thiophenecarboxylate2-(benzoylamino)-N-(3-hydroxypropyl)-4,5-dimethyl-3-thiophenecarboxamide 6-tert-butyl-2-[(2-fluorobenzoyl)amino]-N-(2-hydroxyethyl)-4,5,6,7-tetrahydro-1-benzothiophene-3- carboxamide 2 ethyl5-cyano-6-{[(5-hydroxy-1H-pyrazol-3-yl)methyl]thio}-2-methyl-4-(2-thienyl)-1,4-dihydro-3-pyridinecarboxylate 32-[(5-acetyl-3-cyano-6-methyl-2-pyridinyl)thio]acetamide 43-amino-N-(3-hydroxypropyl)-7,7-dimethyl-7,8-dihydro-5H-thieno[2,3-b]thiopyrano[3,4-e]pyridine-2-carboxamide 51,4-benzothiazin-2-yl)acetamide1,2,3,4-tetrahydro-2-quinoxalinyl)acetamideN-(4-fluorophenyl)succinamide 6 N-(3-methylphenyl)dicarbonimidic diamideN-(2-methoxyphenyl)dicarbonimidic diamideN-(4-chlorophenyl)dicarbonimidic diamideN-(4-methoxyphenyl)dicarbonimidic diamide 7N-(2-hydroxyethyl)-N′-(2-methoxyphenyl)ethanediamideN-(3-fluorophenyl)-N′-(2-hydroxyethyl)ethanediamideN-(2-hydroxyethyl)-N′-(2-methylphenyl)ethanediamideN-1,3-benzodioxol-5-yl-N′-(2-hydroxypropyl)ethanediamideN-(5-chloro-2-methylphenyl)-N′-(3- hydroxypropyl)ethanediamide3,5-dichloro-2-[(2,4-dichlorobenzoyl)amino]-N-(2- hydroxyethyl)benzamide8 1-amino-3-[(2-chloro-4-nitrophenyl)amino]-2-propanol 93-bromo-N-[1-{[(2-hydroxyethyl)amino]carbonyl}-2-(3-nitrophenyl)vinyl]benzamide N-(2-(2-fluorophenyl)-1-{[(3-hydroxypropyl)amino]carbonyl}vinyl)benzamide3-bromo-N-(2-(2-furyl)-1-{[(3-hydroxypropyl)amino]carbonyl}vinyl)benzamide 10 N~2~-acetylarginine 111-[(4-ethoxy-3-methylphenyl)sulfonyl]-N-(2-hydroxyethyl)-3-piperidinecarboxamide N-(3-hydroxypropyl)-1-(2-naphthylsulfonyl)-3-piperidinecarboxamide 12N-[amino(imino)methyl]-4-{[3-(1,3-benzodioxol-5-yl)-3-oxo-1-propen-1-yl]amino}benzenesulfonamide 133-(3-{[(3-hydroxypropyl)amino]sulfonyl}-4,5- dimethoxyphenyl)acrylicacid 14 2-[2-chloro-4-({[2-(2-fluorophenyl)ethyl]amino}methyl)-6-methoxyphenoxy]acetamide 2-(2-chloro-6-methoxy-4-{[(4-pyridinylmethyl)amino]methyl}phenoxy)acetamidehydrochloride2-(2-chloro-6-methoxy-4-{[(2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)amino]methyl}phenoxy)acetamide2-{2-chloro-6-methoxy-4- [(methylamino)methyl]phenoxy}acetamidehydrochloride 2-(2-chloro-6-methoxy-4-{[(3-pyridinylmethyl)amino]methyl}phenoxy)acetamidehydrochloride2-{4-[(1-adamantylamino)methyl]-2-chloro-6- methoxyphenoxy}acetamide2-(2-chloro-6-ethoxy-4-{[(3- pyridinylmethyl)amino]methyl}phenoxy)acetamide hydrochloride 2-{2-chloro-4-[(cyclooctylamino)methyl]-6-methoxyphenoxy}acetamide hydrochloride 2-(2-chloro-6-ethoxy-4-{[(2-thienylmethyl)amino]methyl}phenoxy)acetamide hydrochloride2-(2-bromo-6-methoxy-4-{[(3-pyridinylmethyl)amino]methyl}phenoxy)acetamidehydrochloride 2-(2-chloro-6-methoxy-4-{[(2-thienylmethyl)amino]methyl}phenoxy)acetamide hydrochloride2-(2-chloro-4-{[(2-hydroxypropyl)amino]methyl}-6- methoxyphenoxy)acetamide 2-[2-bromo-6-methoxy-4-({[2-(4-morpholinyl)ethyl]amino}methyl) phenoxy]acetamide dihydrochloride2-{4-[(2-adamantylamino)methyl]-2-chloro-6- methoxyphenoxy}acetamide2-(2-bromo-6-methoxy-4-{[(4-pyridinylmethyl)amino]methyl}phenoxy)acetamidehydrochloride 2-(2-chloro-6-ethoxy-4-formylphenoxy)acetamide2-(2-chloro-6-methoxy-4-{[(tetrahydro-2- furanylmethyl)amino]methyl}phenoxy)acetamide hydrochloride 2-{4-[(benzylamino)methyl]-2-chloro-6-methoxyphenoxy}acetamide hydrochloride2-{2-chloro-4-[(cyclopentylamino)methyl]-6- methoxyphenoxy}acetamidehydrochloride 2-[2-chloro-4-(hydroxymethyl)-6-methoxyphenoxy]acetamide2-(2-chloro-4-{[(4-fluorobenzyl)amino]methyl}-6-methoxyphenoxy)acetamide hydrochloride2-{2-chloro-4-[(cyclopropylamino)methyl]-6- ethoxyphenoxy}acetamidehydrochloride 2-(2-bromo-6-methoxy-4-{[(2-phenylethyl)amino]methyl}phenoxy)acetamide2-{2-chloro-4-[(cyclopropylamino)methyl]-6- methoxyphenoxy}acetamidehydrochloride 2-(2-chloro-6-ethoxy-4-{[(2-furylmethyl)amino]methyl}phenoxy)acetamide hydrochloride2-[2-chloro-4-({[2-(4-fluorophenyl)ethyl]amino}methyl)-6-methoxyphenoxy]acetamide 2-(2-bromo-6-ethoxy-4-{[(2-furylmethyl)amino]methyl}phenoxy)acetamide hydrochloride2-(2-bromo-6-methoxy-4-{[(2-thienylmethyl)amino]methyl}phenoxy)acetamide hydrochloride2-{2-chloro-6-ethoxy-4- [(isopropylamino)methyl]phenoxy}acetamide2-[2-bromo-6-ethoxy-4-(hydroxymethyl)phenoxy]acetamide2-{2-chloro-4-[(cyclohexylamino)methyl]-6- methoxyphenoxy}acetamidehydrochloride 2-(2-bromo-6-methoxy-4-{[(tetrahydro-2-furanylmethyl)amino]methyl} phenoxy)acetamide hydrochloride2-{4-[(butylamino)methyl]-2-chloro-6- methoxyphenoxy}acetamide 152-(4-bromophenoxy)ethanimidamide hydrochloride 162-(1,3-benzodioxol-5-ylmethylene)hydrazinecarboximidamide2-(4-methoxybenzylidene)hydrazinecarboximidamide3-allyl-2-hydroxybenzaldehyde semicarbazone 17N-[(4-methoxybenzyl)oxy]guanidine sulfate 182-(8-quinolinyl)hydrazinecarboxamide 19N-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-1H-pyrazole-5-carboxamide 20 7-methyl-1H-indole-2,3-dione 3-semicarbazone21 N-(aminocarbonyl)-3-oxo-3,4-dihydro-2- quinoxalinecarboxamide 221-benzyl-N-(3-hydroxypropyl)-4-oxo-1,4-dihydropyrido[1,2-a]pyrrolo[2,3-d]pyrimidine-2-carboxamide 232-{[7-(2-hydroxy-3-phenoxypropyl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl]thio}acetamide 246-[(4-bromo-2,3-dimethylphenyl)amino]-2-(3-hydroxypropyl)-5-nitro-1Hbenzo[de]isoquinoline- 1,3(2H)-dione3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propanamide 252-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-N-4H-1,2,4-triazol-4-ylpropanamide 26 N-[amino(imino)methyl]-3,4-dihydro-1(2H)-quinolinecarboximidamide 27 N-(4,6-dimethyl-2-quinazolinyl)guanidineN-(4,6,7-trimethyl-2-quinazolinyl)guanidineN-(4,6,8-trimethyl-2-quinazolinyl)guanidineN-(4,8-dimethyl-2-quinazolinyl)guanidineN-(4,7-dimethyl-2-quinazolinyl)guanidineN-(4-methyl-2-quinazolinyl)guanidineN-(4-methoxy-6-methyl-2-quinazolinyl)guanidine 28N-1,3-benzoxazol-2-ylguanidine 293-amino-N′-(2,4-dichlorobenzylidene)-1H-1,2,4-triazole-5- carbohydrazide30 3-chloro-4-fluoro-N-4H-1,2,4-triazol-4-yl-1-benzothiophene-2-carboxamide5-bromo-2-chloro-N-4H-1,2,4-triazol-4-ylbenzamide2,5-dichloro-N-4H-1,2,4-triazol-4-ylbenzamide oxalate2-chloro-4-methyl-N-4H-1,2,4-triazol-4-ylbenzamide2,4-dimethyl-N-4H-1,2,4-triazol-4-ylbenzamide2,3-dichloro-N-4H-1,2,4-triazol-4-ylbenzamide 31N-[amino(imino)methyl]-4-morpholinecarboximidamide hydrochloride

To demonstrate activity, selected compounds from Table 1 were analyzedfor inhibitory activity. The results of this analysis are presented inTable 2.

TABLE 2 Inhibition Inhibition at 50 μM at 5 μM Compound Structure/Name(%)* (%)*

49.10 17.68 2-(2-chloro-6-ethoxy-4-{[(2-thienylmethyl)amino]methyl}phenoxy) acetamide hydrochloride

45.56 23.05 2-(2-chloro-6-methoxy-4-{[(4-pyridinylmethyl)amino]methyl}phenoxy) acetamide hydrochloride

41.26 10.94 2-(2-chloro-6-methoxy-4-{[(2-thienylmethyl)amino]methyl}phenoxy) acetamide hydrochloride

39.41 19.23 ethyl 2-[(4-amino-4-oxo-2- butenoyl)amino]-4,5-dimethyl-3-thiophenecarboxylate

38.78 14.87 2-(2-chloro-6-ethoxy-4-{[(3-pyridinylmethyl)amino]methyl}phenoxy) acetamide hydrochloride

36.65  8.14 3-bromo-N-[1-{[(2- hydroxyethyl)amino]carbonyl}-2-(3-nitrophenyl)vinyl]benzamide

36.58 18.11 3-(1,3-dioxo-1H-benzo[de]isoquinolin- 2(3H)-yl)propanamide

35.18 13.83 2-{4-[(2-adamantylamino)methyl]-2-chloro-6-methoxyphenoxy}acetamide

31.30 17.00 2-{2-chloro-6-methoxy-4- [(methylamino)methyl]phenoxy}acetamide hydrochloride

31.01 11.80 3-bromo-N-(2-(2-furyl)-1-{[(3-hydroxypropyl)amino]carbonyl}vinyl) benzamide

30.92 16.14 2-(2-bromo-6-ethoxy-4-{[(2-furylmethyl)amino]methyl}phenoxy) acetamide hydrochloride

30.42 13.44 N-(3-hydroxypropyl)-1-(2- naphthylsulfonyl)-3-piperidinecarboxamide

30.23 18.27 2-[2-bromo-6-methoxy-4-({[2-(4-morpholinyl)ethyl]amino}methyl) phenoxy]acetamide dihydrochloride

30.04 11.97 5-bromo-2-chloro-N-4H-1,2,4-triazol-4- ylbenzamide

29.96  4.96 2-{4-[(benzylamino)methyl]-2-chloro-6-methoxyphenoxy}acetamide hydrochloride

29.71  6.67 N-1,3-benzodioxol-5-yl-N′-(2- hydroxypropyl)ethanediamide

29.35 10.94 2-chloro-4-methyl-N-4H-1,2,4-triazol- 4-ylbenzamide

29.33 15.94 2-(2-chloro-6-methoxy-4-{[(2-oxo-2,3-dihydro-1H-benzimidazol-5- yl)amino]methyl}phenoxy)acetamide

27.65 12.21 2-(2-bromo-6-methoxy-4-{[(2-phenylethyl)amino]methyl}phenoxy) acetamide

27.38  4.74 2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-N-4H-1,2,4-triazol-4- ylpropanamide *The observed inhibitionconstant corresponds to 50% of the actual inhibition value. Therefore,if the observed value is 49% the actual value is 98%.

In one embodiment, compounds of the present invention have the structureof Formula I:

wherein R₁ is —H or —CH₃; R₂ is a substituent as found in the compoundsof Tables 1 or 2; and R₃ is a halo group (I, F, Br, Cl).

In another embodiment, compounds of the present invention have thestructure of Formula II:

wherein R₄ is a halo group (I, F, Br, Cl); and R₅ is a substituent asfound in the compounds of Tables 1 or 2.

As demonstrated by the data presented in Table 2, compounds of Table 1,as well as derivatives and analogs thereof (including derivatives andanalogs of Formula I and Formula II), are expected to modulate theactivity of telomerase. Accordingly, the present invention relates toeffector compounds selected for interacting with the FP-pocket oftelomerase. In particular embodiments, a compound of the invention isselected from the group listed in Table 1. In another embodiment, one ormore of the compounds listed in Table 1 serve as lead compounds fordesigning or generating derivatives or analogs which are more potent,more specific, less toxic and more effective than known inhibitors oftelomerase or the lead compound. Derivatives or analogs can also be lesspotent but have a longer half-life in vivo and/or in vitro and thereforebe more effective at modulating telomerase activity in vivo and/or invitro for prolonged periods of time.

Derivatives or analogs of the compounds disclosed herein typicallycontain the essential elements of the parent compound, but have had oneor more atoms (e.g., halo, lower alkyl, hydroxyl, amino, thiol, ornitro), or group of atoms (e.g., amide, aryl, heteroaryl, allyl, orpropargyl), replaced or added. Such replacements or substitutions caninclude substituent R groups and/or atoms of the core structure, e.g.,replacing a carbon with a heteroatom such as a nitrogen, oxygen, orsulfur. In this regard, the compounds disclosed herein serve as leadcompounds for creating a family of derivatives or analogs of use ininhibiting or activating telomerase to, e.g., alter lifespan orproliferative capacity of a cell.

The term “effector” refers to an agonist, antagonist, ligand or otheragent that affects the activity of telomerase. Effectors that bind theFP-pocket of telomerase and, e.g., occlude the active site of telomeraseact as effective telomerase-specific inhibitors, whereas effectors inclose proximity to the active site of the enzyme without occluding theactive site act as effective telomerase-specific activators.

Molecules or compounds of the present invention were selected forinteracting with the FP-pocket of telomerase. Such selection was basedupon various heterogeneous interactions between the compound andtelomerase including, but not limited to van der Waals contacts,hydrogen bonding, ionic interactions, polar contacts, or combinationsthereof. In this regard, the terms “bind,” “binding,” “interact,” or“interacting” are used interchangeably herein to describe the physicalinteractions between amino acid residues of the FP-pocket of telomeraseand effectors thereof. In general, the molecules of the invention wereselected for interacting with 2, 3, 4, 5, 6 or more of the amino acidresidues of the FP-pocket of telomerase.

In the context of the present invention, telomerase refers to a familyof enzymes which maintain telomere ends by addition of the telomererepeat TTAGGG. Telomerases are described, e.g., by Nakamura, et al.(1997) Science 277(5328):955-9 and O′Reilly, et al. (1999) Curr. Opin.Struct. Biol. 9(1):56-65. Exemplary telomerase enzymes, as well asconserved motifs and structures thereof, are set forth herein in SEQ IDNOs:1-8 (FIGS. 2A-2C). In addition, full-length sequences for telomeraseenzymes are known in the art under GENBANK Accession Nos. AAC39140(Tetrahymena thermophila), NP_(—)197187 (Arabidopsis thaliana),NP_(—)937983 (Homo sapiens), CAA18391 (Schizosaccharomyces pombe),NP_(—)033380 (Mus musculus), NP_(—)013422 (Saccharomyces cerevisiae),AAC39163 (Oxytricha trifallax), CAE75641 (Euplotes aediculatus) andNP_(—)001035796 (Tribolium castaneum). Reference to telomerase refers toallelic and synthetic variants of telomerase, as well as fragments oftelomerase which fold to form the FP-pocket.

The effector activity of compounds of the invention toward telomerasecan be confirmed using any conventional assay for measuring telomeraseactivity. For example, such assays include combining telomerase orsuitable fragments of telomerase with or without substrates or cofactors(e.g., TER, complementary DNA, nucleotides, or Mg) in solution anddetermining whether a test compound can block or enhance telomeraseactivity. Such activities of telomerase include telomerase catalyticactivity (which may be either processive or non-processive activity);telomerase processivity; conventional reverse transcriptase activity;nucleolytic activity; primer or substrate (telomere or synthetictelomerase substrate or primer) binding activity; dNTP binding activity;RNA (i.e., TER) binding activity; and protein binding activity (e.g.,binding to telomerase-associated proteins, telomere-binding proteins, orto a protein-telomeric DNA complex). See, e.g., assays disclosed in U.S.Pat. No. 7,262,288.

Telomerase catalytic activity is intended to encompass the ability oftelomerase to extend a DNA primer that functions as a telomerasesubstrate by adding a partial, one, or more than one repeat of asequence (e.g., TTAGGG) encoded by a template nucleic acid (e.g., TER).This activity may be processive or non-processive. Processive activityoccurs when a telomerase RNP adds multiple repeats to a primer ortelomerase before the DNA is released by the enzyme complex.Non-processive activity occurs when telomerase adds a partial, or onlyone, repeat to a primer and is then released. In vivo, however, anon-processive reaction could add multiple repeats by successive roundsof association, extension, and dissociation. This can occur in vitro aswell, but it is not typically observed in standard assays due to thevastly large molar excess of primer over telomerase in standard assayconditions. Conventional assays for determining telomerase catalyticactivity are disclosed, for example, in Morin (1989) Cell 59:521; Morin(1997) Eur. J. Cancer 33:750; U.S. Pat. No. 5,629,154; WO 97/15687; WO95/13381; Krupp, et al. (1997) Nucleic Acids Res. 25:919; Wright, et al.(1995) Nuc. Acids Res. 23:3794; Tatematsu, et al. (1996) Oncogene13:2265.

Telomerase conventional reverse transcriptase activity is described in,e.g., Morin (1997) supra, and Spence, et al. (1995) Science 267:988.Because telomerase contains conserved amino acid motifs that arerequired for reverse transcriptase catalytic activity, telomerase hasthe ability to transcribe certain exogenous (e.g., non-TER) RNAs. Aconventional RT assay measures the ability of the enzyme to transcribean RNA template by extending an annealed DNA primer. Reversetranscriptase activity can be measured in numerous ways known in theart, for example, by monitoring the size increase of a labeled nucleicacid primer (e.g., RNA or DNA), or incorporation of a labeled dNTP. See,e.g., Ausubel, et al. (1989) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y.

Because telomerase specifically associates with TER, it can beappreciated that the DNA primer/RNA template for a conventional RT assaycan be modified to have characteristics related to TER and/or atelomeric DNA primer. For example, the RNA can have the sequence(CCCTAA)_(n), where n is at least 1, or at least 3, or at least 10 ormore. In one embodiment, the (CCCTAA)_(n) region is at or near the 5′terminus of the RNA (similar to the 5′ locations of template regions intelomerase RNAs). Similarly, the DNA primer may have a 3′ terminus thatcontains portions of the TTAGGG telomere sequence, for exampleX_(n)TTAG, X_(n)AGGG, etc., where X is a non-telomeric sequence and n is6-30. In another embodiment, the DNA primer has a 5′ terminus that isnon-complementary to the RNA template, such that when the primer isannealed to the RNA, the 5′ terminus of the primer remains unbound.Additional modifications of standard reverse transcription assays thatmay be applied to the methods of the invention are known in the art.

Telomerase nucleolytic activity is described in, e.g., Morin (1997)supra and Collins & Grieder (1993) Genes Dev. 7:1364. Telomerasepreferentially removes nucleotides, usually only one, from the 3′ end ofan oligonucleotide when the 3′ end of the DNA is positioned at the 5′boundary of the DNA template sequence, in humans and Tetrahymena, thisnucleotide is the first G of the telomeric repeat (TTAGG in humans).Telomerase preferentially removes G residues but has nucleolyticactivity against other nucleotides. This activity can be monitored usingconventional methods known in the art.

Telomerase primer (telomere) binding activity is described in, e.g.,Morin (1997) supra; Collins, et al. (1995) Cell 81:677; Harrington, etal. (1995) J. Biol. Chem. 270:8893. There are several ways of assayingprimer binding activity; however, a step common to most methods isincubation of a labeled DNA primer with telomerase or telomerase/TERunder appropriate binding conditions. Also, most methods employ a meansof separating unbound DNA from protein-bound DNA. Such methods caninclude, e.g., gel-shift assays or matrix binding assays. The DNA primercan be any DNA with an affinity for telomerase, such as, for example, atelomeric DNA primer like (TTAGGG)_(n), where n could be 1-10 and istypically 3-5. The 3′ and 5′ termini can end in any location of therepeat sequence. The primer can also have 5′ or 3′ extensions ofnon-telomeric DNA that could facilitate labeling or detection. Theprimer can also be derivatized, e.g., to facilitate detection orisolation.

Telomerase dNTP binding activity is described in, e.g., Morin (1997)supra and Spence, et al. (1995) supra. Telomerase requires dNTPs tosynthesize DNA. The telomerase protein has a nucleotide binding activityand can be assayed for dNTP binding in a manner similar to othernucleotide binding proteins (Kantrowitz, et al. (1980) Trends Biochem.Sci. 5:124). Typically, binding of a labeled dNTP or dNTP analog can bemonitored as is known in the art for non-telomerase RT proteins.

Telomerase RNA (i.e., TER) binding activity is described in, e.g., Morin(1997) supra; Harrington, et al. (1997) Science 275:973; Collins, et al.(1995) Cell 81:677. The RNA binding activity of a telomerase protein ofthe invention may be assayed in a manner similar to the DNA primerbinding assay described supra, using a labeled RNA probe. Methods forseparating bound and unbound RNA and for detecting RNA are well known inthe art and can be applied to the activity assays of the invention in amanner similar to that described for the DNA primer binding assay. TheRNA can be full length TER, fragments of TER or other RNAs demonstratedto have an affinity for telomerase or TRBD. See U.S. Pat. No. 5,583,016and WO 96/40868.

In addition to determining either RNA or DNA binding, assays monitoringbinding of the RNA-templating region and the complementary telomeric DNAsequence can also be carried out. By way of illustration, Example 3describes the use of fluorescence polarization to determine binding ofcompounds to TERT protein.

To further evaluate the efficacy of a compound identified using themethod of the invention, one of skill will appreciate that a modelsystem of any particular disease or disorder involving telomerase can beutilized to evaluate the adsorption, distribution, metabolism andexcretion of a compound as well as its potential toxicity in acute,sub-chronic and chronic studies. For example, the effector or modulatorycompound can be tested in an assay for replicative lifespan inSaccharomyces cerevisiae (Jarolim, et al. (2004) FEMS Yeast Res.5(2):169-77). See also, McChesney, et al. (2005) Zebrafish 1(4):349-355and Nasir, et al. (2001) Neoplasia 3(4):351-359, which describe marinemammal and dog tissue model systems for analyzing telomerase activity.

Compounds disclosed herein find application in a method for modulating(i.e., blocking or inhibiting, or enhancing or activating) telomerase.Such a method involves contacting a telomerase either in vitro or invivo with an effective amount of a compound of the invention so that theactivity of telomerase is modulated. An effective amount of an effectoror modulatory compound is an amount which reduces or increases theactivity of the telomerase by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%or 100% when compared to telomerase not contacted with the compound.Such activity can be monitored by enzymatic assays detecting activity ofthe telomerase or by monitoring the expression or activity of proteinswhich are known to be associated with or regulated by telomerase.

Modulation of telomerase activity finds application in selectivelyanalyzing telomerase signaling events in model systems as well as inpreventing or treating diseases, conditions, and disorders involving orassociated with telomerase activity or reduction or lack thereof. Theselection of the compound for use in preventing or treating a particulardisease or disorder will be dependent upon the particular disease ordisorder. For example, human telomerase is involved in cancer andtherefore a compound which inhibits telomerase is useful in theprevention or treatment of cancer including solid tumors (e.g.,adenocarcinoma of the breast, prostate, and colon; melanoma; non-smallcell lung; glioma; as well as bone, breast, digestive system,colorectal, liver, pancreatic, pituitary, testicular, orbital, head andneck, central nervous system, acoustic, pelvic, respiratory tract, andurogenital neoplasms) and leukemias (e.g., B-cell, mixed-cell,null-cell, T-cell, T-cell chronic, lymphocytic acute, lymphocyticchronic, mast-cell, and myeloid). Cancer cells (e.g., malignant tumorcells) that express telomerase activity (telomerase-positive cells) canbe mortalized by decreasing or inhibiting the endogenous telomeraseactivity. Moreover, because telomerase levels correlate with diseasecharacteristics such as metastatic potential (e.g., U.S. Pat. Nos.5,639,613; 5,648,215; 5,489,508; Pandita, et al. (1996) Proc. Am. Ass.Cancer Res. 37:559), any reduction in telomerase activity could reducethe aggressive nature of a cancer to a more manageable disease state(increasing the efficacy of traditional interventions).

By way of illustration, Example 4 describes cell-based assays and animalmodel systems which are useful for assessing the inhibition of tumorcell growth by one or more compounds of the invention. Another usefulmethod for assessing anticancer activities of compounds of the inventioninvolves the multiple-human cancer cell line screening assays run by theNational Cancer Institute (see, e.g., Boyd (1989) in Cancer: Principlesand Practice of Oncology Updates, DeVita et al., eds, pp. 1-12). Thisscreening panel, which contains approximately 60 different human cancercell lines, is a useful indicator of in vivo antitumor activity for abroad variety of tumor types (Greyer, et al. (1992) Seminars Oncol.19:622; Monks, et al. (1991) Natl. Cancer Inst. 83:757-766), such asleukemia, non-small cell lung, colon, melanoma, ovarian, renal,prostate, and breast cancers. Antitumor activities can be expressed interms of ED₅₀ (or GI₅₀), where ED₅₀ is the molar concentration ofcompound effective to reduce cell growth by 50%. Compounds with lowerED₅₀ values tend to have greater anticancer activities than compoundswith higher ED₅₀ values.

Upon the confirmation of a compound's potential activity in one or morein vitro assays, further evaluation is typically conducted in vivo inlaboratory animals, for example, measuring reduction of lung nodulemetastases in mice with B16 melanoma (e.g., Schuchter, et al. (1991)Cancer Res. 51:682-687). The efficacy of a compound of the inventioneither alone or as a drug combination chemotherapy can also beevaluated, for example, using the human B-CLL xenograft model in mice(e.g., Mohammad, et al. (1996) Leukemia 10:130-137). Such assaystypically involve injecting primary tumor cells or a tumor cell lineinto immune compromised mice (e.g., a SCID mouse or other suitableanimal) and allowing the tumor to grow. Mice carrying the tumors arethen treated with the compound of interest and tumor size is measured tofollow the effect of the treatment. Alternatively, the compound ofinterest is administered prior to injection of tumor cells to evaluatetumor prevention. Ultimately, the safety and efficacy of compounds ofthe invention are evaluated in human clinical trials.

Compounds that activate or stimulate telomerase activity findapplication in methods for treating or preventing a disease or conditionwherein telomerase activity is lacking or reduced, e.g., conditionsrelating to the proliferative capacity of cells, conditions resultingfrom cell damage or death, or conditions associated with cellularsenescence. As such stimulatory compounds find use in methods fordecreasing the rate of senescence of a subject, e.g., after onset ofsenescence; for extending the lifespan of a subject; or for treating orpreventing a disease or condition relating to lifespan. Certain diseasesof aging are characterized by cell senescence-associated changes due toreduced telomere length (compared to younger cells), resulting from theabsence (or much lower levels) of telomerase activity in the cell.Telomerase activity and telomere length can be increased by, forexample, increasing the activity of telomerase in the cell. A partiallisting of conditions associated with cellular senescence in whichincreased telomerase activity can be therapeutic includes Alzheimer'sdisease, Parkinson's disease, Huntington's disease, and stroke;age-related diseases of the integument such as dermal atrophy,elastolysis and skin wrinkling, graying of hair and hair loss, chronicskin ulcers, and age-related impairment of wound healing; degenerativejoint disease; osteoporosis; age-related immune system impairment (e.g.,involving cells such as B and T lymphocytes, monocytes, neutrophils,eosinophils, basophils, NK cells and their respective progenitors);age-related diseases of the vascular system; diabetes; and age-relatedmacular degeneration. Moreover, telomerase activators can be used toincrease the proliferative capacity of a cell or in cellimmortalization, e.g., to produce new cell lines (e.g., most humansomatic cells).

Prevention or treatment typically involves administering to a subject inneed of treatment a pharmaceutical composition containing an effectiveof a compound of the invention. In most cases this will be a humanbeing, but treatment of agricultural animals, e.g., livestock andpoultry, and companion animals, e.g., dogs, cats and horses, isexpressly covered herein. The selection of the dosage or effectiveamount of a compound is that which has the desired outcome ofpreventing, reducing or reversing at least one sign or symptom of thedisease or disorder being treated. Methods for treating cancer and othertelomerase-related diseases in humans are described in U.S. Pat. Nos.5,489,508, 5,639,613, and 5,645,986. By way of illustration, a subjectwith cancer (including, e.g., carcinomas, melanomas, sarcomas, lymphomasand leukaemias) can experience unexplained weight loss, fatigue, fever,pain, skin changes, sores that do not heal, thickening or lump in breastor other parts of the body, or a nagging cough or hoarseness, whereintreatment with a compound of the invention can prevent, reduce, orreverse one or more of these symptoms.

Pharmaceutical compositions can be in the form of pharmaceuticallyacceptable salts and complexes and can be provided in a pharmaceuticallyacceptable carrier and at an appropriate dose. Such pharmaceuticalcompositions can be prepared by methods and contain carriers which arewell-known in the art. A generally recognized compendium of such methodsand ingredients is Remington: The Science and Practice of Pharmacy,Alfonso R. Gennaro, editor, 20th ed. Lippincott Williams & Wilkins:Philadelphia, Pa., 2000. A pharmaceutically-acceptable carrier,composition or vehicle, such as a liquid or solid filler, diluent,excipient, or solvent encapsulating material, is involved in carrying ortransporting the subject compound from one organ, or portion of thebody, to another organ, or portion of the body. Each carrier must beacceptable in the sense of being compatible with the other ingredientsof the formulation and not injurious to the subject being treated.

Examples of materials which can serve as pharmaceutically acceptablecarriers include sugars, such as lactose, glucose and sucrose; starches,such as corn starch and potato starch; cellulose, and its derivatives,such as sodium carboxymethyl cellulose, ethyl cellulose and celluloseacetate; powdered tragacanth; malt; gelatin; talc; excipients, such ascocoa butter and suppository waxes; oils, such as peanut oil, cottonseedoil, safflower oil, sesame oil, olive oil, corn oil and soybean oil;glycols, such as propylene glycol; polyols, such as glycerin, sorbitol,mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyllaurate; agar; buffering agents, such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters,polycarbonates and/or polyanhydrides; and other non-toxic compatiblesubstances employed in pharmaceutical formulations. Wetting agents,emulsifiers and lubricants, such as sodium lauryl sulfate and magnesiumstearate, as well as coloring agents, release agents, coating agents,sweetening, flavoring and perfuming agents, preservatives andantioxidants can also be present in the compositions.

Compounds of the present invention can be used alone or in combinationwith other agents, such as cancer therapeutic agents, in the treatmentof disease. Thus, in particular embodiments, the present inventionembraces combining an effective amount of a compound of the inventionwith one or more cancer therapeutic agents. A cancer therapeutic is usedin the conventional sense to refer to chemotherapeutic andradiotherapeutic agents that control or kill malignant or cancer cells.Such agents include, but are not limited to, conventional cytostatic orcytotoxic agents and immune modulators; radiation therapy;molecule-targeted drugs; or any kind of immune therapy includingvaccination, lymphocytes, dendritic cells; or a combination thereof.

More particularly, cancer chemotherapeutic agents refer to agents thatinduce apoptosis and/or impair mitosis of rapidly dividing cells. Cancerchemotherapeutic agents of use in accordance with the present inventioninclude, but are not limited to, alkylating agents (e.g., cisplatin,carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, andchlorambucil); antimetabolites (e.g., azathioprine and mercaptopurine);anthracyclines; plant products including vinca alkaloids (e.g.,Vincristine, Vinblastine, Vinorelbine, and Vindesine) and taxanes (e.g.,paclitaxel); and topoisomerase inhibitors (e.g., amsacrine, irinotecan,topotecan, etoposide, etoposide phosphate, and teniposide), which affectcell division or DNA synthesis and/or function; as well as monoclonalantibodies (e.g., trastuzumab, cetuximab, rituximab, and Bevacizumab)and tyrosine kinase inhibitors such as imatinib mesylate, which directlytarget a molecular abnormality in certain types of cancer (e.g., chronicmyelogenous leukemia, gastrointestinal stromal tumors).

A radiotherapeutic agent refers to an agent the produces ionizingradiation that damages cellular DNA. Radiotherapy is conventionallyprovided as external beam radiotherapy (EBRT or XBRT) or teletherapy,brachytherapy or sealed source radiotherapy, and systemic radioisotopetherapy or unsealed source radiotherapy. The differences relate to theposition of the radiation source; external is outside the body,brachytherapy uses sealed radioactive sources placed precisely in thearea under treatment, and systemic radioisotopes are given by infusionor oral ingestion. In this regard, when used in the context of acomposition of the present invention, a radiotherapeutic is intended tomain a radioactive agent used in brachytherapy. When used in the contextof the methods of the present invention, a cancer therapeutic includesall forms of radiotherapy routinely used in the art.

The selection of one or more appropriate cancer therapeutics for use inthe composition and methods of the invention can be carried out theskilled practitioner based upon various factors including the conditionof the patient, the mode of administration, and the type of cancer beingtreated. Moreover, the combination therapy can be included in the sameor multiple pharmaceutical compositions. In addition, the individualdrugs can be administered simultaneously or consecutively (e.g.,immediately following or within an hour, day, or month of each other).

The compositions of the present invention can be administeredparenterally (for example, by intravenous, intraperitoneal, subcutaneousor intramuscular injection), topically (including buccal andsublingual), orally, intranasally, intravaginally, or rectally accordingto standard medical practices.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion or metabolism of the particularcompound being employed, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularcompound employed, the age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of a compound at levels lower than that required in order toachieve the desired therapeutic effect and gradually increase the dosageuntil the desired effect is achieved. This is considered to be withinthe skill of the artisan and one can review the existing literature on aspecific compound or similar compounds to determine optimal dosing.

The invention is described in greater detail by the followingnon-limiting examples.

Example 1 Structure of T. castaneum TERT in Complex with DNA Substrate

Protein Expression and Purification. The synthetic gene of T. castaneumfull-length TERT was cloned into a modified version of the pET28b vectorcontaining a cleavable hexahistidine tag at its N-terminus. The proteinwas over-expressed in E. coli BL21 (pLysS) at 30° C. for 4 hours. Thecells were lysed by sonication in 50 mM Tris-HCl, 10% glycerol, 0.5 MKCl, 5 mM β-mercaproethanol, and 1 mM PMSF, pH 7.5 on ice. The proteinwas first purified over a Ni-NTA column followed by TEV cleavage of thehexahistidine tag overnight at 4° C. The TERT/TEV mixture was dialyzedto remove the excess imidazole and the protein was further purified overa second Ni-NTA column that was used to remove all his-tagged products.The Ni-NTA flow through was then passed over a POROS-HS column(Perceptive Biosystems) to remove any trace amounts of proteincontaminants. At this stage the protein was more than 99% pure. Theprotein was finally purified over a SEPHEDEX-S200 sizing columnpre-equilibrated with 50 mM Tris-HCl, 10% glycerol, 0.5 M KCl, and 1 mMTris(2-Carboxyethyl) phosphine (TCEP), pH 7.5 to remove any TERTaggregates and the protein was concentrated to 10 mg/ml using an AMICON30K cutoff (MILLIPORE) and stored at 4° C. for subsequent studies. Stockprotein was dialyzed in 10 mM Tris-HCl, 200 mM KCl, 1 mM TCEP, pH 7.5prior to crystallization trials.

Protein Crystallization and Data Collection. Initial crystal trials ofthe protein alone did not produce crystals. Co-crystallization of theprotein with single stranded telomeric DNA ((TCAGG)₃) produced tworod-like crystal forms one of which belongs to the orthorhombic spacegroup P2₁2₁2₁ and diffracted to 2.71 Å and the other to the hexagonalspace group P6₁ and diffracted to 3.25 Å resolution. The protein nucleicacid mix was prepared prior to setting crystal trials by mixing onevolume of dialyzed protein with 1.2-fold excess of the DNA substrate.Both crystal forms where grown by the vapor diffusion, sitting dropmethod by mixing on volume of the protein-DNA mix with one volume ofreservoir solution. Orthorhombic crystals where grown in the presence of50 mM HEPES, (pH 7.0) and 1.5 M NaNO₃ while hexagonal crystals grew inthe presence of 100 mM Tris (pH 8.0) and 2 M (NH₄)₂SO₄ and both at roomtemperature. Orthorhombic crystals were harvested into cryoprotectantsolution that contained 50 mM HEPES (pH 7.0), 25% glycerol, 1.7 M NaNO₃,0.2 M KCl and 1 mM TCEP and were flash frozen in liquid nitrogen.Hexagonal crystals were harvested into cryoprotectant solution thatcontained 100 mM Tris (pH 8.0), 25% glycerol, 2 M (NH₄)₂SO₄, 0.2 M KCland 1 mM TCEP and were also flash frozen in liquid nitrogen. Data werecollected at the NSLS, beam line X6A and processed with HKL-2000 (Minor(1997) Methods in Enzymology: Macromolecular Crystallography, part A276:307-326) (Table 3). Both crystal forms contain a dimer in theasymmetric unit.

TABLE 3 Native 1 Native 2 Hg1 Hg2 Data collection Space group P2₁2₁2₁P6₁ P2₁2₁2₁ P2₁2₁2₁ Cell dimensions a, b, c (Å) 85.0420, 200.0670,86.7165, 86.9260, 122.6570, 200.0670, 123.3500, 123.4100, 212.406096.4100 211.4530 211.4160 Resolution (Å)   40-2.70   40-3.25  40-3.5  40-3.5 (2.78-2.71)* (3.32-3.25) (3.69-3.5)  (3.69-3.5) R_(sym) orR_(merge) 10.7 14.9 14.5 16.1 (48.1) (42.6) (41.7) (43.7) I/σI 9.3 6.47.0 7.3 (1.7) (2.4) (3.5) (3.6) Completeness 96.97 98.85 85.7 93.8 (%)(95.84) (98.1) (83.1) (94.2) Redundancy 4.2 2.8 4.7 5.3 (4.2) (2.5)(4.8) (5.3) Refinement Resolution (Å)  20-2.71   20-3.25 No. reflections56173 32773 R_(work)/R_(free) 23.8/27.7 24.3/29.6 No. atoms Protein 49824982 Water 358 77 B-factors Protein 52.5 37.8 Water 41.3 26.5 R.m.sdeviations Bond lengths 0.007 0.006 (Å) Bond angles (°) 0.848 0.735Ramachandran plot (%) Most favored 83.3 86.4 Allowed 15.2 11.5Generously 1.4 1.7 allowed Disallowed 0.2 0.4 *Highest resolution shellis shown in parenthesis.

Structure Determination and Refinement. Initial phases for theorthorhombic crystals were obtained using the method of singleisomorphous replacement with anomalous signal (SIRAS) using two datasetscollected from two different mercury (CH₃HgCl) derivatized crystals attwo different wavelengths (Hg1-1.00850 Å; Hg2-1.00800 Å) (Table 3). Thederivatives were prepared by soaking the crystals with 5 mM methylmercury chloride (CH₃HgCl) for 15 minutes. Initially, twelve heavy atomsites were located using SOLVE (Terwilliger (2003) Methods Enzymol.374:22-37) and refined and new phases calculated with MLPHARE(Collaborative Computational Project 4 (1994) Acta Crystallogr. D50:760-763). MLPHARE improved phases were used to identify the remainingheavy atom sites (twenty two in total) by calculating an anomalousdifference map to 3.5 Å resolution. MLPHARE phases obtained using allthe heavy atom sites where then used in DM with two-fold NCS and phaseextension using the high-resolution (2.71 Å) dataset collected, at1.00800 Å wavelength, to calculate starting experimental maps. Thesemaps were sufficiently good for model building which was carried out inCOOT (Emsley & Cowtan (2004) Acta Crystallogr D Biol Crystallogr60:2126-32). The electron density map revealed clear density for all 596residues of the protein. However, density for the nucleic acid substratein the structure was not observed. The model was refined using bothCNS-SOLVE (Brunger, et al. (1998) Acta Crystallogr D Biol Crystallogr54:905-21) and REFMAC5 (Murshudov, et al. (1997) Acta Crystallogr D BiolCrystallogr 53:240-55). The last cycles of refinement were carried outwith TLS restraints as implemented in REFMAC5 (Table 3). The P2₁2₁2₁refined model was used to solve the structure of the TERT crystallizedin the P6₁ crystal form (data collected at 0.97980 Å wavelength) bymolecular replacement with PHASER (Potterton, et al. (2003) ActaCrystallogr D Biol Crystallogr 59:1131-7).

Architecture of the TERT Structure. The structure of the full-lengthcatalytic subunit of the T. castaneum active telomerase, TERT, wasdetermined to 2.71 Å resolution. As indicated, there was a dimer in theasymmetric unit (AU), however the protein alone was clearly monomeric insolution as indicated by gel filtration and dynamic light scattering,indicating that the dimer observed in the crystal was the result ofcrystal packing. This was further supported by the fact that a differentcrystal form (Table 3) of the same protein also contained a dimer in theAU of different configuration. It is worth noting that the TERT fromthis organism does not contain a TEN domain, a low conservation regionof telomerase (FIG. 1B).

The TERT structure is composed of three distinct domains, a TER-bindingdomain (TRBD), the reverse transcriptase (RT) domain, and the C-terminalextension thought to represent the putative “thumb” domain of TERT(FIGS. 1A And 1B). As indicated herein, the TRBD is mostly helical andcontains an indentation on its surface formed by two conserved motifs(CP and T) which bind double- and single-stranded RNA, respectively, andhas been defined as the template boundary element of the RNA substrateof telomerase, TER. Structural comparison of the TRBD domain from T.castaneum with that of the structure from T. thermophila shows that thetwo structures are similar (RMSD 2.7 Å), indicating a high degree ofstructural conservation between these domains across organisms ofdiverse phylogenetic groups.

The RT domain is a mix of α-helices and β-strands organized into twosubdomains that are most similar to the “fingers” and “palm” subdomainsof retroviral reverse transcriptases such as HIV reverse transcriptase(PDB code ID 1N5Y; Sarafianos, et al. (2002) EMBO J. 21:6614-24), viralRNA polymerases such as hepatitis C viral polymerase (Code ID 2BRL DiMarco, et al. (2005) J. Biol. Chem. 280:29765-70) and B-family DNApolymerases such as RB69 (PDB Code ID 1WAF; Wang, et al. (1997) Cell89:1087-99), and contain key signature motifs that are hallmarks ofthese families of proteins (Lingner, et al. (1997) Science 276:561-7)(FIGS. 2A-2C). Structural comparison of TERT with the HIV RTs, showsthat the “fingers” subdomain of TERT (i.e., motifs 1 and 2) are arrangedin the open configuration with respect to the “palm” subdomain (i.e.,motifs A, B′, C, D, and E), which is in good agreement with theconformation adopted by HIV RTs in the absence of bound nucleotide andnucleic acid substrates (Ding, et al. (1998) J. Mol. Biol.284:1095-111). One striking difference between the putative “palm”domain of TERT and that HIV reverse transcriptases is a long insertionbetween motifs A and B′ of TERT referred to as the IFD motif that isrequired for telomerase processivity (Lue, et al. (2003) Mol. Cell.Biol. 23:8440-9). In the TERT structure, the IFD insertion is composedof two anti-parallel α-helices (α13 and α14) located on the outsideperiphery of the ring and at the interface of the “fingers” and the“palm” subdomains. These two helices are almost in parallel positionwith the central axis of the plane of the ring and make extensivecontacts with helices α10 and α15 and play an important role in thestructural organization of this part of the RT domain. A similarstructural arrangement is also present in viral polymerases, and theequivalent of helix α10 in these structures is involved in directcontacts with the nucleic acid substrate (Ferrer-Orta, et al. (2004) J.Biol. Chem. 279:47212-21).

In contrast to the RT domain, the C-terminal extension is an elongatedhelical bundle that contains several surface exposed, long loops. Asearch in the protein structure database using the software SSM(Krissinel & Henrick (2004) Acta Cryst. D60:2256-2268; Krissinel (2007)Bioinformatics 23:717-723) produced no structural homologues suggestingthat the CTE domain of telomerase adopts a novel fold. Structuralcomparison of TERT with the HIV RT, the viral RNA polymerases andB-family DNA polymerases places the “thumb” domain of these enzymes andthe CTE domain of TERT in the same spatial position with respect to the“fingers” and “palm” subdomains, indicating that the CTE domain oftelomerase is the “thumb” domain of the enzyme, a finding that is ingood agreement with previous biochemical studies (Hossain, et al. (2002)J. Biol. Chem. 277:36174-80).

TERT domain organization brings the TRBD and “thumb” domains, whichconstitute the terminal domains of the molecule, together, anarrangement that leads to the formation of a ring-like structure that isreminiscent of the shape of a donut (FIG. 1C). Several lines of evidenceindicate that the domain organization of the TERT structure presentedherein is biologically relevant. First, the domains of four TERTmonomers observed in two different crystal forms (two in each asymmetricunit) are organized the same (average RMSD=0.76 Å between all fourmonomers). Second, contacts between the N- and C-terminal domains ofTERT are extensive (1677 Å²) and largely hydrophobic in nature involvingamino acid residues Tyr4, Lys76, Thr79, Glu84, Ser81, His87, Asn142,His144, Glu145, Tyr411, His 415, Phe417, Trp420, Phe422, Ile426, Phe434,Thr487, Ser488, Phe489, and Arg592. This observation is in agreementwith previous biochemical studies (Arai, et al. (2002) J. Biol. Chem.277:8538-44). Third, TERT domain organization is similar to that of thepolymerase domain (p66 minus the RNase H domain) of its closesthomologue, HIV reverse transcriptase (Sarafianos, et al. (2002) supra),the viral RNA polymerases (Di Marco, et al. (2005) supra) and theB-family DNA polymerases and in particular RB69 (Wang, et al. (1997)supra). The arrangement of the TERT domains creates a hole in theinterior of the particle that is ˜26 Å wide and ˜21 Å deep, sufficientto accommodate double-stranded nucleic acids approximately seven toeight bases long, which is in good agreement with existing biochemicaldata (Forstemann & Lingner (2005) EMBO Rep. 6:361-6; Hammond & Cech(1998) Biochemistry 37:5162-72).

The TERT Ring Binds Double-Stranded Nucleic Acid. To understand how theTERT ring associates with RNA/DNA to form a functional elongationcomplex, a double-stranded nucleic acid was modeled into the interiorusing the HIV reverse transcriptase—DNA complex (Sarafianos, et al.(2002) supra), TERT's closest structural homologue. The TERT-RNA/DNAmodel immediately showed some striking features that supported the modelof TERT-nucleic acid associations. The hole of the TERT ring and wherethe nucleic acid heteroduplex was projected to bind was lined withseveral key signature motifs that are hallmarks of this family ofpolymerases and have been implicated in nucleic acid association,nucleotide binding and DNA synthesis. Moreover, the organization ofthese motifs resulted in the formation of a spiral in the interior ofthe ring that resembled the geometry of the backbone of double-strandednucleic acid. Several of the motifs, identified as contact points withthe DNA substrate, were formed partly by positively charged residues,the side chains of which extended toward the center of the ring and werepoised for direct contact with the backbone of the DNA substrate. Forexample, the side chain, of the highly conserved K210 that forms part ofhelix α10, is within coordinating distance of the backbone of themodeled DNA thus providing the stability required for a functionaltelomerase enzyme. Helix α10 lies in the upper segment of the RT domainand faces the interior of the ring. The location and stabilization ofthis helix is heavily influenced by its extensive contacts with the IFDmotif implicated in telomerase processivity (Lue, et al. (2003) Mol.Cell. Biol. 23:8440-9). Disruption of the IFD contacts with helix α10through deletion or mutations of this motif would lead to displacementof helix α10 from its current location, which would in turn effectDNA-binding and telomerase function.

Structural elements of the “thumb” domain that localized to the interiorof the ring also made several contacts with the modeled DNA substrate.In particular, the loop (“thumb” loop) that connects the “palm” to the“thumb” domain and constitutes an extension of the E motif also known asthe “primer grip” region of telomerase, preserves to a remarkabledegree, the geometry of the backbone of double stranded nucleic acid.The side chains of several lysines (e.g., Lys406, Lys416, Lys418) andasparagines (e.g., Asn423) that formed part of this loop extended towardthe center of the TERT molecule and were within coordinating distance ofthe backbone of modeled double-stranded nucleic acid. Of particularinterest was Lys406. This lysine was located in proximity of motif E andits side chain extended toward the nucleic acid heteroduplex and waspoised for direct contacts with the backbone of the nucleotides locatedat the 3′ end of the incoming DNA primer. It is therefore possible thatthe side chain of this lysine together with motif E help facilitateplacement of the 3′-end of the incoming DNA substrate at the active siteof the enzyme during telomere elongation. Sequence alignments of the“thumb” domain of TERTs from a wide spectrum of phylogenetic groupsshowed that the residues predicted to contact the DNA substrate arealways polar (FIGS. 2A-2C). Another feature of the “thumb” domain thatsupported double-stranded nucleic acid binding was helix α19, a 3¹⁰helix (“thumb” 3¹⁰ helix) that extended into the interior of the ringand appeared to dock itself into the minor groove of the modeleddouble-stranded nucleic acid thus facilitating RNA/DNA hybrid bindingand stabilization. Deletion or mutation of the corresponding residues inboth yeast and human TERT results in sever loss of TERT processivityclearly indicating the important role of this motif in TERT function(Hossain, et al. (2002) J. Biol. Chem. 277:36174-80; Huard, et al.(2003) Nucleic Acids Res. 31:4059-70; Banik, et al. Mol. Cell. Biol.22:6234-46).

The Active Site of TERT and Nucleotide Binding. The T. castaneum TERTstructure presented herein was crystallized in the absence of nucleotidesubstrates and magnesium, however, the location and organization ofTERT's active site and nucleotide binding pocket was determined on thebasis of existing biochemical data (Lingner, et al. (1997) supra) andstructural comparison with the polymerase domain of its closesthomologue, the HIV reverse transcriptase (Das, et al. (2007) J. Mol.Biol. 365:77-89). The TERT active site is composed of three invariantaspartic acids (Asp251, Asp343 and Asp344) that form part of motifs Aand C, two short loops located on the “palm” subdomain, and adjacent tothe “fingers” of TERT. Structural comparison of TERT with HIV reversetranscriptases, as well as RNA and DNA polymerases showed a high degreeof similarity between the active sites of these families of proteinsindicating that telomerase also employs a two-metal mechanism forcatalysis. Alanine mutants of these TERT aspartic acids resulted incomplete loss of TERT activity indicating the essential role of theseresidues in telomerase function (Lingner, et al. (1997) supra).

The telomerase nucleotide binding pocket is located at the interface ofthe “fingers” and “palm” subdomains of TERT and is composed of conservedresidues that form motifs 1, 2, A, C, B′ and D implicated in templateand nucleotide binding (Bosoy & Lue (2001) J. Biol. Chem. 276:46305-12;Haering, et al. (2000) Proc. Natl. Acad. Sci. USA 97:6367-72).Structural comparisons of TERT with viral HIV reverse transcriptasesbound to ATP (Das, et al. (2007) supra) supports nucleotide substrate inthis location. Two highly conserved, surface-exposed residues Tyr256 andVal342 of motifs A and C, respectively, form a hydrophobic pocketadjacent to and above the three catalytic aspartates and couldaccommodate the base of the nucleotide substrate. Binding of thenucleotide in this oily pocket places the triphospate moiety inproximity of the active site of the enzyme for coordination with one ofthe Mg²⁺ ions while it positions the ribose group within coordinatingdistance of an invariant glutamine (Gln308) that forms part of motif B′thought to be an important determinant of substrate specificity (Smith,et al. (2006) J. Virol. 80:7169-78). Protein contacts with thetriphospate moiety of the nucleotide are mediated by motif D, a longloop, located beneath the active site of the enzyme. In particular, theside chain of the invariant Lys372 is within coordinating distance ofthe γ-phosphate of the nucleotide an interaction that most likely helpsposition and stabilize the triphosphate group during catalysis. The sidechains of the highly conserved Lys189 and Arg194 of motifs 1 and 2,which together form a long β-hairpin that forms part of the “fingers”subdomain, are also within coordinating distance of the both the sugarand triphosphate moieties of the modeled nucleotide. Contacts witheither or both the sugar moiety and the triphosphate of the nucleotidesubstrate would facilitate nucleotide binding and positioning forcoordination to the 3′-end of the incoming DNA primer.

TRBD Facilitates Template Positioning at the Active Site of TERT. Aswith most DNA and RNA polymerases, nucleic acid synthesis by telomeraserequires pairing of the templating region (usually seven to eight basesor more) of TER with the incoming DNA primer (Lee & Blackburn (1993)Mol. Cell. Biol. 13:6586-99). TRBD-RT domain organization forms a deepcavity on the surface of the protein that spans the entire width of thewall of the molecule, forming a gap that allows entry into the hole ofthe ring from its side. The arrangement of this cavity with respect tothe central hole of the ring provides an elegant mechanism for placementof the RNA template, upon TERT-TER assembly, in the interior of the ringand where the enzyme's active site is located. Of particularsignificance is the arrangement of the β-hairpin that forms part of theT-motif. This hairpin extends from the RNA-binding pocket and makesextensive contacts with the “thumb” loop and motifs 1 and 2. Contactsbetween this hairpin and both the “fingers” and the “thumb” domainsplace the opening of the TRBD pocket that faces the interior of the ringin proximity to the active site of the enzyme. It is therefore likelythat this β-hairpin acts as an allosteric effector switch that couplesRNA-binding in the interior of the ring and placement of the RNAtemplate at the active site of the enzyme. Placement of the templateinto the interior of the molecule would facilitate its pairing with theincoming DNA substrate, which together would form the RNA/DNA hybridrequired for telomere elongation. RNA/DNA pairing is a prerequisite oftelomere synthesis in that it brings the 3′-end of the incoming DNAprimer in proximity to the active site of the enzyme for nucleotideaddition while the RNA component of the heteroduplex provides thetemplate for the faithful addition of identical repeats of DNA at theends of chromosomes. Strikingly, modeling of the RNA/DNA heteroduplex inthe interior of the TERT ring places the 5′-end of the RNA substrate atthe entry of the RNA-binding pocket and where TERT is expected toassociate with TER while it places the 3′-end of the incoming DNA primerat the active site of TERT providing a snapshot of the organization of afunctional telomerase elongation complex.

Example 2 Structure of T. castaneum Telomerase Catalytic Subunit TERTBinding to RNA Template and Telomeric DNA

Methods for Expressing and Purifying Proteins. Wild-type and mutant(Asp251Ala) T. castaneum, full length TERT proteins were overexpressedand purified as described herein with subtle modifications thatincreased protein yield. Specifically, the proteins were over-expressedin E. coli Rosetta (DE3) pLysS (Novagen) at 30° C. for 5 hours with slowshaking (shaker/incubator setting, 120 rpm). Stock protein (10 mg/ml)was dialyzed in 10 mM Tris-HCl, 100 mM KCl, 1 mM TCEP, pH 7.5 prior tocrystallization trials.

Methods for Preparing T. castaneum Extracts and Isolating RNA. Twenty T.castaneum larvae or pupae were ground in liquid N₂, homogenized with 200μl of extraction buffer (25 mM Tris-HCl, 5 mM β-mercaptoethanol (β-ME),1 mM EGTA, 0.1 mM benzamidine, 200 mM KCl, 10% (w/v) glycerol, 10 mMimidazole and RNasin (PROMEGA, Madison, Wis.), pH 7.5) and placed on icefor 30 minutes. After the homogenate was centrifuged at 12,000 g at 4°C. for 20 minutes, the supernatant was collected, flush frozen in liquidnitrogen and stored at −80° C. before use. The total RNA was thenextracted from the T. castaneum homogenate using the RNEASY Protect MiniKit (QIAGEN, Valencia, Calif.).

Methods for In Vitro Reconstitution of T. castaneum Telomerase. Thetelomerase RNA of T. castaneum had not been previously identified.Therefore, total RNA was isolated from beetle larvae and used with therecombinant TERT to assemble the telomerase complex in vitro. Twenty μgof the his-tagged TERT (25 mM Tris, 200 mM KCl, 10% (w/v) glycerol, 5 mM(3-ME and 10 mM imidazole, pH 7.5) was mixed with 50 μl of T. castaneumlarvae total RNA and the two were incubated in T. castaneum lysate fortwo hours at room temperature in the presence of RNasin. The telomerasecomplex was then purified over a Ni-NTA column and tested for activityusing a modified TRAP assay (Sasaki & Fujiwara (2000) Eur. J. Biochem.267:3025-3031) as described herein.

Telomerase Repeat Amplification Protocol (TRAP) Assays. The activity ofthe in vitro reconstituted T. castaneum telomerase was tested using thefollowing TRAP assay. The telomerase elongation step was carried out ina 50 μl reaction mixture composed of 20 mM Tris-HCl (pH 8.3), 7.5 mMMgCl₂, 63 mM KCl, 0.05% (w/v) TWEEN 20, 1 mM EGTA, 0.01% (w/v) BSA, 0.5mM of each dNTP, 1 μM DNA primer (5′-aag ccg tcg agc aga gtc-3′; SEQ IDNO:9) (Tcas-TS)) and 4 μg of Ni-NTA purified, T. castaneum telomerase.After incubation at 30° C. for 60 minutes, each reaction mixture wasphenol-chloroform extracted and precipitated with ethanol. Each samplewas re-suspended in 50 μl of PCR reaction buffer (10 mM Tris/HCl (pH8.0), 50 mM KCl, 2 mM MgCl₂, 100 μM dNTPs (dATP dTTP dGTP), 10 μM[³²P]dCTP (80 Ci per mmol), 1 μM Tcas-CX primer (5′-gtg tga cct gac ctgacc-3′; SEQ ID NO:10) and HOTSTARTAQ DNA polymerase (QIAGEN). The PCRproducts were resolved on Tris-borate-EDTA (TBE)-polyacrylamide gels.The presence of multiple telomeric repeats (TCAGG)_(n) was alsoconfirmed by subcloning and sequencing the TRAP products.

RNA-DNA Hairpins. The RNA-DNA hairpins tested for TERT binding, activityand subsequently for structural studies are shown in Table 4. All threehairpins contain the putative RNA-templating region(5′-rCrUrGrArCrCrU-3′) (one and a half times the telomeric repeat of T.castaneum, TCAGG) and the complementary telomeric DNA sequence(5′-GTCAGGT-3′). The RNA-templating region and the DNA complement wereconnected for stability with an RNA-DNA linker (loop). The hairpins weredesigned to contain a 5′-RNA overhang so that the telomerase complexcould be trapped in its replication state upon co-crystallization withthe complementary non-hydrolysable nucleotide(s). The only differencebetween the three hairpins is the length of the linker.

TABLE 4 Hairpin Sequence (5′->3′) SEQ ID NO: 21mer_(r)C_(r)U_(r)G_(r)A_(r)C_(r)C_(r)U _(r)G_(r)A_(r)CTTCGGTCAGGT 11 17mer_(r)C_(r)U_(r)G_(r)A_(r)C_(r)C_(r)U _(r)GTTCGCAGGT 12 15mer_(r)C_(r)U_(r)G_(r)A_(r)C_(r)C_(r)U TTCG AGGT 13 *Underlined sequencesrepresent linker sequences. Bold sequences form the loop of the hairpin.

TERT, Reverse Transcriptase (RT) Assays. Standard reverse transcriptaseassays were carried out using the recombinant T. castaneum TERT and theRNA-DNA hairpin (Table 4) to test TERT's ability to replicate the end ofthe DNA substrate that includes part of the RNA-DNA hairpin. RT assayswere carried out in telomerase buffer (50 mM Tris-HCl, 100 mM KCl, 1.25mM MgCl₂, 5 mM DTT, 5% (w/v) glycerol, pH 8 at room temperature), 100 μMdNTPs (dATP dTTP dGTP), 10 μM [³²P]dCTP (80 Ci per mmol), 5 μM RNA-DNAhairpin and 1 μM recombinant TERT. The samples were incubated for twohours at room temperature, then phenol/chloroform extracted and ethanolprecipitated. The DNA pellet was re-suspended in a solution composed of90% (w/v) formamide and 10% (w/v) glycerol and the sample was run on a12% (w/v) polyacrylamide-7 M urea gel in 1×TBE at 220V for 70 minutes at4° C.

Protein Crystallization and Data Collection. The binary complex wasprepared by adding to the dialyzed protein 1.2 molar excess nucleic acid(RNA-DNA hairpin purchased from Integrated DNA Technologies), 5 mMdNTPaS (Jena Biosciences GmbH) and 5 mM MgCl₂. Crystals of themonoclinic space group P2₁ that diffracted to 2.7 Å resolution appearedin three days and grew to final size in two weeks. Crystals were grownby the vapor diffusion, sitting drop method by mixing one volume of theternary complex with one volume of reservoir solution containing 0.1 MHEPES, (pH 7.5) and 12% 1,6-hexanediol or PEG 4K and 0.2 M KCl. Crystalswere transferred into cryoprotectant solution that contained 0.1 M HEPES(pH 7.5), 15% (w/v) 1,6-hexanediol or PEG 4K, 15% (w/v) glycerol, 0.2 MKCl and 1 mM TCEP and were harvested by flash freezing in liquidnitrogen. Data were collected at the NSLS, beam line X25 and processedwith MOSFILM as implemented in WEDGER-ELVES (Holton & Alber (2004) Proc.Natl. Acad. Sci. USA 101:1537-1542) (Table 5). There is one monomer inthe asymmetric unit.

TABLE 5 TERT Complex Data Collection Space group P2₁ Cell dimensions a,b, c (Å) 77.2 52.8 101.6 α, β, γ (°) 90 101.9 90 Resolution (Å)   20-2.7 (2.85-2.70)* R_(sym) or R_(merge) 11.4 (45.8) I/σI 7.9 (2.3)Completeness (%) 93.7 (96.1) Redundancy 2.8 (2.5) Refinement Resolution(Å) 20-2.7 No. Reflections 19815 R_(work)/R_(free) 24.2/28.7 Number ofatoms 4982 Protein 4982 Ligand/Ion 496/1  Water 54 B-factors Protein 45Ligand/Ion 37 Water 21 R.m.s. Deviations Bond lengths (Å) 0.006 Bondangles (°) 0.887 *Number of crystals used - one. *Values in parenthesesare for highest-resolution shell.

Structure Determination and Refinement. Phases where calculated bymolecular replacement (MR) using PHASER (Potterton, et al. (2003) ActaCrystallogr D Biol Crystallogr 59:1131-1137) as implemented in CCP4 suitof programs using the substrate-free TERT structure described herein asa search model. Maps calculated after one cycle of refinement byCNS-SOLVE revealed clear 2fo-fc density for all 596 residues of TERT andfo-fc density for the nucleic acid substrate at 3.0 σ contour level.Model building was carried out in COOT (Emsley & Cowtan (2004) ActaCrystallogr D Biol Crystallogr 60:2126-2132) and the model was refinedusing both CNS-SOLVE (Brunger, et al. (1998) Acta Crystallogr. D Biol.Crystallogr. 54:905-921) and REFMAC5 (Murshudov, et al. (1997) ActaCrystallogr. D Biol. Crystallogr. 53:240-255). The last cycles ofrefinement were carried out with TLS restraints as implemented inREFMAC5. The structure was refined to good stereochemistry with 84.3,14.3 and 1.4 of the residues in the most favorable, additional allowedand generously allowed of the Ramachandran plot, respectively.

TERT Structure Overview. The full length (FIG. 1B), active, T.castaneum, TERT was co-crystallized with an RNA-DNA hairpin containingthe putative RNA templating region (5′-rCrUrGrArCrCrU-3′) and thecomplementary telomeric DNA (5′-GTCAGGT-3′) joined together with a shortRNA-DNA linker (Table 4). It is worth noting that the T. castaneum TERTlacks the TEN domain, required for activity and processivity in severaleukaryotic telomerase genes, including human (Wyatt, et al. (2009) PLoSOne 4:e7176) and Tetrahymena thermophila (Zaug, et al. (2008) Nat.Struct. Mol. Biol. 15:870-872; Finger & Bryan (2008) Nucleic Acids Res.36:1260-1272). The absence of the TEN domain from T. castaneum couldexplain the reduced activity observed for this enzyme when compared tothose containing this domain. The RNA component of T. castaneumtelomerase has not been previously identified. Therefore, the followinginformation was used to predict its templating region: The RNAtemplating region of telomerase is usually one and a half telomericrepeats (Lee & Blackburn (1993) Mol. Cell. Biol. 13: 6586-6599; Lingner,et al. (1994) Genes Dev. 8: 1984-1998; Shippen-Lentz & Blackburn (1990)Science 247: 546-552) and this sequence is known for many organisms(telomerase database). For example the mammalian telomerase templatingregion is “CUAACCCU” and the telomeric repeat is “TTAGGG”. The telomericrepeat for T. castaneum is “TCAGG” (Osanai, et al. (2006) Gene376:281-89; Richards, et al. (2008) Nature 452:949-955). The RNA-DNAhairpin was designed to contain a three-nucleotide overhang at the5′-end of the RNA template, so that the enzyme could be trapped in itscatalytic state upon co-crystallization of the protein-nucleic acidassembly with Mg⁺⁺ ions and non-hydrolysable nucleotides. In an effortto identify a hairpin suitable for crystallographic studies, a number ofRNA-DNA hairpins were screened (15mer, 18mer and 21mer; Table 4) wherethe templating region and the complementary DNA sequence were kept thesame but the length of the linker was changed. Although, all threehairpins tested had the same binding affinity for TERT and reversetranscriptase activity, only the 21mer was amenable to crystallographicstudies. It was noted that in the structure, the hairpin linker extendsout of the TERT ring and is only involved in crystal contacts withadjacent molecules. The crystals were also grown in the presence of theslowly hydrolysable nucleotide analogues dNTPαS and Mg⁺⁺ ions.

The structure was solved at 2.7 Å resolution using the method ofmolecular replacement with the substrate-free TERT described herein as asearch model. All of the TERT molecule and the RNA-DNA hybrid wereinterpretable in electron density maps. Unexpectedly, there was extradensity for three nucleotides at the 3′-end of the telomeric DNA,indicating that TERT had extended the 3′-end of the DNA substrate in thecrystallization drop. There was no evidence for nucleotide at the activesite of the enzyme, which was partially occupied by the nucleotidelocated at the 3′-end of the DNA substrate. Several lines of evidenceindicate that the association of the RNA templating region and the DNAsubstrate with TERT in the structure presented here are biologicallyrelevant. First, TERT is an active polymerase in the presence of thenucleic acid substrate used in this study both by standard reversetranscriptase assays and in the crystallization drop. Second, the RNAtemplate makes contacts with conserved motifs that are hallmarks of thisfamily of enzymes. Third, contacts between TERT and the RNA templateposition the solvent accessible bases adjacent to and above the activesite of the enzyme for nucleotide binding thus facilitating selectivity.Moreover, TERT-RNA associations position the 5′-end of the RNA-templateat the entry of the TRBD RNA-binding pocket and where the templateboundary element located upstream of the RNA-template in many organismsis thought to bind. Fourth, interactions between the DNA substrate andthe primer grip region (a characteristic shared among telomerase and HIVRTs) place the 3′-end of the DNA substrate at the active site of theenzyme where it is accessible for nucleotide addition. Fifth,TERT-nucleic acid associations are strikingly similar to those observedof HIV reverse transcriptase, TERT's closest structural homologue.

The four major TERT domains: the RNA-binding domain (TRBD); the fingersdomain, implicated in nucleotide binding and processivity (Bosoy & Lue(2001) J. Biol. Chem. 276:46305-46312); the palm domain, which containsthe active site of the enzyme; and the thumb domain, implicated in DNAbinding and processivity (Hossain, et al. (2002) J. Biol. Chem.277:36174-80), were organized into a ring configuration similar to thatobserved for the substrate-free enzyme described herein (FIG. 1C). Thearrangement of the TERT domains created a highly positively chargedcavity in the interior of the TERT ring, which was 22 Å wide and 21 Ådeep and could accommodate seven bases of double stranded nucleic acid.Within this cavity binds one molecule of the RNA-DNA hybrid, whichassembles together, via Watson-Crick base pairing into a helicalstructure similar to both the DNA-DNA and RNA-DNA structure bound to HIVreverse transcriptase (Huang, et al. (1998) Science 282:1669-75;Sarafianos, et al. (2001) EMBO J. 20:1449-61).

TERT Nucleic Acid Associations. Structure analysis indicated thatinteractions between the protein and the RNA templating region weremediated by the fingers, the palm and thumb domains. The 5′-end RNAcytosine (rC1) and uracil (rU2) were located at the interface of thefingers and palm domains and were involved in a network of interactionswith conserved residues (FIG. 3) of motifs 2 and B′ both of which arelocated in proximity of the active site of the enzyme. In particular,the 2′-OH and the base carbonyl of rC1 is within hydrogen bondingdistance of the backbone carbonyls of Val197 of motif 2 and Gly309 ofmotif B′ while the pyrimidine base sits over the otherwise solventexposed, hydrophobic side chain of the conserved Ile196 that also formspart of motif 2 (FIG. 4). Contacts between rU2 and the protein aremediated by the short aliphatic side chain of Pro311 and the ribosegroup (FIG. 4). Interactions between rC1 and rU2 with motifs 2 and B′place the cytosine base in proximity of the active site of the enzyme,where it is well-positioned for Watson-Crick base-pairing with theincoming nucleotide substrate. Stabilization and placement of the 5′-endbases of the templating region above the active site of the enzyme wasin large part mediated by the interactions of the remaining fiveribonucleotides with the incoming DNA primer. Limited contacts betweenthis part of the RNA and the protein were mediated via a water molecule(Wat18) which coordinates the 2′-OH of guanosine (rG3) with the backboneof helix α15 (FIG. 4). Notably, the structural organization of helix α15was influenced by the IFD motif, a long insertion, composed of twohelices (α13 and α14), between motifs A and B′, which explains whymutations in this motif lead to loss of telomerase function (Lue, et al.(2003) Mol. Cell. Biol. 23:8440-49).

Contacts between TERT and the DNA substrate were mediated in large partvia backbone interactions with the thumb loop and helix. The thumbhelix, as described herein, sits in the minor groove of the RNA-DNAheteroduplex, making extensive contacts with the phosphodiester backboneand the ribose groups of the RNA-DNA hybrid. The mode of action of thethumb helix of telomerase appears to be similar to that proposed for theequivalent helix (helix H) of retroviral reverse transcriptases(Jacobo-Molina, et al. (1993) Proc. Natl. Acad. Sci. USA 90:6320-24;Kohlstaedt, et al. (1992) Science 256:1783-1790). Another conservedelement of the thumb domain, known as the thumb loop, runs almostparallel to the curvature of the DNA primer and the two are involved ina network of backbone and solvent mediated interactions (FIG. 5).Interactions between the DNA and the thumb loop included the side chainsof Lys416 and Asn423, both of which extended toward the center of thering and were within hydrogen bonding distance of the DNA backbone.Contacts between the thumb domain and the DNA position the nucleotideslocated at its 3′-end within coordinating distance of the primer gripregion (motif E), a short, rigid loop located at the interface of thepalm and thumb domains, and in proximity of the active site of theenzyme (FIG. 6). The backbone of the tip of this loop formed by theconserved residues Cys390 and Gly391 abuts the ribose group of C22 andthis interaction guides the 3′-end DNA nucleotides toward the activesite of the enzyme (FIG. 6). The active site of the enzyme, and wherethe incoming nucleotide is projected to bind, is partially occupied bythe nucleotide (G24) located at the 3′-end of the DNA (FIG. 6). Theribose group, and to a certain extend the guanosine base of G24, whichmakes Watson-Crick pairing interactions with the rC1 located at the5′-end of the RNA template, sit in a well-defined hydrophobic pocketformed by the side chains of the invariant Tyr256, Gln308 and conservedVal342 of motifs A, B′ and C respectively, while the α-phosphate iscoordinated by the magnesium ion occupying the active site aspartates(FIG. 6). The important role of Val342 in telomerase selectivity hasbeen shown for the human telomerase holoenzyme (Drosopoulos & Prasad(2007) Nucleic Acids Res. 35:1155-1168).

TERT Domain Rearrangements Upon Nucleic Acid Binding. Comparisons of thenucleic acid bound and substrate-free TERT structures indicate that TERTnucleic acid associations induce small rigid-body changes in orientationbetween subunits of the enzyme that lead to a 3.5 Å decrease of thediameter of the interior cavity of the ring. The decrease in thediameter of the central cavity arises from a 6° inward rotation togetherwith a 3.5 Å translation, of the thumb domain with respect to thefingers and palm domains. Translation of the thumb domain toward thecenter of the ring is accompanied by the TRBD, which is also shifted 3.5Å toward the finger domain creating a more narrow RNA-binding pocketthan the substrate-free enzyme. The role of this subtle structuralrearrangement may have implications for TERT association with thefull-length RNA, TER.

Common Aspects of Substrate Binding Between TERT and HIV RTs. It hasbeen postulated that telomerase uses a mechanism of DNA replication thatresembles that of other retroviral reverse transcriptases, a suggestionsupported by the structure of the TERT-nucleic acid complex presentedhere. Structural comparison of the RNA-DNA bound TERT and HIV RT (PDBID: 1HYS36) shows a striking similarity in the overall domainorganization and nucleic acid binding between the two structures. LikeHIV RTs, telomerase-dependent telomere replication requires pairing ofthe templating region with the incoming DNA primer and placement of the3′-end of the DNA into the enzyme's active site for nucleotide addition.Moreover, TERT- or HIV RT-nucleic acid associations are accompanied bydomain rearrangements that facilitate the formation of a tight,catalytic, protein nucleic acid assembly and positioning of the DNA3′-end at the active site of the enzyme for catalysis (Kohlstaedt, etal. (1992) supra; Rodgers, et al. (1995) Proc. Natl. Acad. Sci. USA92:1222-1226; Steitz (1997) Harvey Lect. 93:75-93). Contacts between theprotein and the RNA templating region are specific and involvetelomerase key signature motifs (motif 2 and B′ of the fingers and palmdomains, respectively) that are hallmarks of these families of enzymesand are required for positioning of the solvent accessible bases of theRNA template in proximity of the active site for nucleotide binding andselectivity. Contacts between the protein and the DNA substrate aremediated by the thumb domain and despite the lack of sequence homologyin this region between the two families of enzymes, the mode of actionof the thumb helix of telomerase is similar to that proposed for helix Hof HIV RTs (Jacobo-Molina, et al. (1993) supra; Kohlstaedt, et al.(1992) supra; Beese, et al. (1993) Science 260: 352-355). Placement ofthe DNA 3′-end at the active site of the enzyme is further facilitatedby the primer grip region another highly conserved motif between TERTand HIV RTs44 (FIG. 6).

Although the enzyme was not trapped in its catalytic state, thepartially occupied active site of the enzyme, formed by a number ofinvariant (Asp251, Tyr256, Gln308, Asp343, Asp344) or highly conservedresidues (Val342), by the nucleotide G24 located at the 3′-end of theDNA (FIG. 6) suggests the mechanism of nucleotide binding andselectivity of telomerase during the replication process. In a similarmanner, the invariant Tyr256 and Gln308 are also present in HIV RTswhere, like in TERT, they are involved in nucleotide binding andselectivity through positioning for interactions with the templatingregion (Huang, et al. (1998) supra; Cases-Gonzalez, et al. (2000) J.Biol. Chem. 275:19759-19767), further supporting common mechanisticaspects of DNA replication between these families of enzymes.

TERT Rigid Conformational Changes and Function. Domain re-organization,upon nucleic acid binding, are a common feature of RNA-, DNA-polymerasesand retroviral reverse transcriptases, and are geared toward theformation of a tight, catalytic protein nucleic acid assembly andpositioning of the DNA 3′-end at the active site of the enzyme forcatalysis (Kohlstaedt, et al. (1992) supra; Rodgers, et al. (1995)supra; Steitz (1997) supra). Unlike HIV RTs, telomerase appears toexist, at least in the absence of the full-length integral RNAcomponent, in a closed ring configuration, an arrangement mediated byextensive contacts between the TRBD and the thumb domains. Comparison ofthe nucleic acid bound and substrate-free TERT structures indicate thatTERT nucleic acid associations induce subtle, rigid-body changes inorientation between subunits of the enzyme that lead to a 3.5 Å decreaseof the diameter of the interior cavity of the ring. These observationswere unexpected because in most polymerases, including the HIV RT, thefingers and thumb domains undergo significant conformational changesrequired for substrate binding and function (Steitz (1997) supra; Ding,et al. (1998) J. Mol. Biol. 284:1095-1111; Steitz (1999) J. Biol. Chem.274:17395-98). For example, the fingers domain, which is known to bindand position the nucleotide at the active site of the enzyme, undergoessignificant conformational changes referred to as the open and closedstates (Ding, et al. (1998) supra). It is therefore possible that theinteractions between the TRBD and the thumb domains lock the fingersdomain in place, thus preventing the conformational rearrangementsobserved in other polymerases, which would indicate the possibility of apreformed active site. A preformed active site has been observed for theHepatitis C viral RNA polymerase (NS5B) (Bressanelli, et al. (2002) J.Virol. 76:3482-92), a close structural homologue of TERT but also theY-family DNA polymerases (Ling, et al. (2001) Cell 107:91-102). Anotherpossibility is that the substrate-free TERT enzyme was trapped in theclosed fingers conformation. Assuming the later is true, significantmovement of the fingers domain of TERT would most likely require thatthe TRBD and the thumb domains are splayed apart. Contacts between theTRBD and the thumb domain are extensive and would require significantenergy to force them apart. This could be accomplished by accessoryproteins that possibly act in a similar manner to that of the slidingclamp loader of DNA polymerases (Jeruzalmi, et al. (2002) Curr. Opin.Struct. Biol. 12:217-224).

Repeat Addition Processivity. Telomerase, unlike most polymerases, hasthe ability to add multiple identical repeats of DNA to the ends ofchromosomes, known as repeat addition processivity. This uniquecharacteristic of telomerase has been attributed in part to theassociation of the N-terminal portion of TERT with TER and the telomericoverhang as well as the IFD motif (Lue, et al. (2003) supra). The TENdomain, present in several organisms, and its weak interaction with theDNA substrate is thought to be a determinant of repeat additionprocessivity (Wyatt, et al. (2007) Mol. Cell. Biol. 27:3226-40;Moriarty, et al. (2004) Mol. Cell. Biol. 24:3720-33), while mostrecently the TRBD and its stable association with TER has also beenshown to be involved in this process (see data presented herein). In thecomplex presented here, the RNA does not directly engage the RNA-bindingpocket of TRBD. The structure shows that TERT-RNA contacts position the5′-end of the templating region at the entry of the RNA-binding pocketof TRBD. This arrangement would place the template boundary element(Chen &7 Greider (2003) Genes Dev. 17:2747-2752; Lai, et al. (2002)Genes Dev. 16:415-20; Tzfati, et al. (2000) Science 288:863-867) presentin most organisms or the short oligonucleotide overhang of rodent TER51within the RNA binding pocket of TRBD. The stable association of TERwith the TRBD would force the enzyme to stall when reaching thenucleotide located at the 5′-end of the RNA template thus preventingreplication beyond this point. Stalling of the enzyme for extendedperiods would lead to destabilization and dissociation of the RNA-DNAheteroduplex and initiation of another round of telomere replication.

Collectively, the data presented here supports common mechanisticaspects of substrate binding and DNA replication between telomerase andHIV reverse transcriptases, indicating an evolutionary link betweenthese families of enzymes. It also provides novel insights into thebasic mechanisms of telomere replication and length homeostasis bytelomerase. Moreover, the structure presented here provides a detailedpicture of the physical contacts between TERT and its nucleic acidsubstrates, information of use in the design of small moleculeinhibitors of telomerase having therapeutic value in the treatment ofcancer and other diseases associated with cellular aging.

Example 3 Fluorescence Polarization Binding Assay

The TERT protein was expressed and purified as described herein. Proteinwas concentrated to 4.72 mg ml-1 using an AMICON 30K cutoff filter(Millipore) and stored at −80° C. for binding assays.

For the binding assay, TERT protein was diluted to a 500 nMconcentration in a solution of 50 mM Tris-HCl, pH 7.5, 200 mM KCl, and20% glycerol. A 1.2-fold molar excess of the Chim21m RNA-DNA hybrid(Integrated DNA Technologies), containing the template for telomereextension (5′-rCrUrG rArCrC rUrGrA rCTT CGG TCA GGT-3′; SEQ ID NO:11),was then added to these samples and kept on ice. The cyanine 5-dCTP(Perkin Elmer) for the binding study was made up to a 20 nMconcentration in sterile, RNase, DNase free water with 40 nM MgCl₂.

Compound screens were carried out as 30.5 μl reactions in a 384-well,round bottom plate (Thermo). Fifteen μl of TERT-Chim21m complex wasfirst added to each well. Then 0.5 μl of library compound (ChemBridge)was added to this solution. Finally, 15 μl of the nucleotide-MgCl2sample was added, and the solution mixed to provide final concentrationsof 250 nM protein-nucleic acid, 10 nM cyanine 5-dCTP, and 20 nM MgCl₂.Final compound concentrations were 50 μM and 5 μM. All additions werecompleted using a Janus 96/384 Modular Dispensing Tool (Perkin Elmer),performed in triplicate, and equilibrated for 40 minutes. Fluorescencepolarization was measured at λ_(ex)=620 nm and λ_(em)=688 nm on anEnVision Xcite Multilabel plate reader (Perkin Elmer). Polarization wascalculated using the standard equation: P=(V−H)/(V+H), where P denotespolarization, V denotes vertical emission intensity, and H denoteshorizontal emission intensity.

Example 4 Efficacy of Telomerase Inhibitors

Novel telomerase inhibitors of the instant invention can be analyzed ina variety of systems. The compounds can be assessed in definedwell-known model systems used to assess cellular permeability, toxicity,and pharmacodynamic effects. These assays include both cell-based andanimal based assays.

Cell-Based Assay. Cells from a P388 cell line (CellGate, Inc.,Sunnyvale, Calif.) or human malignant melanoma cell line SK-MEL-2 aregrown in RPMI 1640 cell medium containing fetal calf serum (10%),L-glutamine, penicillin, streptomycin and are split twice weekly. Allcompounds are first diluted with DMSO. Later serial dilutions are donewith a phosphate-buffered saline solution. All dilutions are done inglass vials and the final DMSO concentration is generally below 0.5% byvolume. Final two-fold dilutions are done in a 96-well plate using cellmedia so that each well contains 50 μL. All compounds are assayed overmultiple concentrations. Cell concentration is measured using ahemacytometer and the final cell concentration is adjusted to about1×10⁴ cells/mL with cell medium. The resulting solution of cells (50 μL)is then added to each well and the plates are incubated for 5 days in a37° C., 5% CO₂, humidified incubator. MTT solution(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, 10 μL) isthen added to each well and the plates are re-incubated under identicalconditions for 2 hours. To each well is then added acidified isopropanol(150 μL of i-PrOH solution containing 0.05 N HCl) and mixed thoroughly.The plates are then scanned at 595 nm and the absorbances are read(Wallac Victor 1420 Multilabel Counter). The resulting data is thenanalyzed to determine an ED₅₀ value. Compounds that kill cancer cells,but fail to kill normal cells, find application in the prevention ortreatment of cancer.

Mouse Ovarian Carcinoma Zenograft Model. Compounds of the invention areevaluated in an ovarian carcinoma xenograft model of cancer, based onthat described by Davis, et al. ((1993) Cancer Research 53:2087-2091).This model, in brief, involves inoculating female nu/nu mice with 1×10⁹OVCAR3-icr cells into the peritoneal cavity. One or more test compoundsare administered, e.g., prior to or after tumor cell injection, by theoral route as a suspension in 1% methyl cellulose or intraperitoneallyas a suspension in phosphate-buffered saline in 0.01% TWEEN-20. At theconclusion of the experiment (4-5 weeks) the number of peritoneal cellsare counted and any solid tumor deposits weighed. In some experimentstumor development is monitored by measurement of tumor specificantigens.

Rat Mammary Carcinoma Model. Compounds of the invention are evaluated ina HOSP.1 rat mammary carcinoma model of cancer (Eccles, et al. (1995)Cancer Res. 56:2815-2822). This model involves the intravenousinoculation of 2×10⁴ tumor cells into the jugular vein of female CBH/cbirats. One or more test compounds are administered, e.g., prior to orafter tumor cell injection, by the oral route as a suspension in 1%methyl cellulose or intraperitoneally as a suspension inphosphate-buffered saline and 0.01% TWEEN-20. At the conclusion of theexperiment (4-5 weeks) the animals are killed, the lungs are removed andindividual tumors counted after 20 hours fixation in Methacarn.

Mouse B16 Melanoma Model. The anti-metastatic potential of compounds ofthe invention is evaluated in a B16 melanoma model in C57BL/6. Mice areinjected intravenously with 2×10⁵ B16/F10 murine tumor cells harvestedfrom in vitro cultures. Inhibitors are administered by the oral route asa suspension in 1% methyl cellulose or intraperitoneally as a suspensionin phosphate-buffered saline pH 7.2 and 0.01% TWEEN-20. Mice are killed14 days after cell inoculation and the lungs removed and weighed priorto fixing in Bouin's solution. The number of colonies present on thesurface of each set of lungs is then counted.

1. A compound selected for interacting with the Fingers-Palm pocket oftelomerase.
 2. The compound of claim 1, wherein the compound has astructure comprising:


3. The compound of claim 1, wherein the compound is selected from thegroup of compounds listed in Table
 1. 4. A pharmaceutical compositioncomprising the compound of claim 1 in admixture with a pharmaceuticallyacceptable carrier.
 5. The pharmaceutical composition of claim 4,further comprising a cancer therapeutic agent.
 6. The pharmaceuticalcomposition of claim 5, wherein the cancer therapeutic agent comprises achemotherapeutic agent or radiotherapeutic agent.
 7. A method formodulating telomerase activity comprising contacting telomerase with acompound of claim 1 thereby modulating the activity of telomerase. 8.The method of claim 7, wherein the compound inhibits telomeraseactivity.
 9. The method of claim 8, wherein the compound has a structurecomprising:


10. The compound of claim 8, wherein the compound is selected from thegroup of compounds listed in Table
 2. 11. The method of claim 7, whereinthe compound stimulates telomerase activity.
 12. A method for preventingor treating a disease or disorder associated with telomerase comprisingadministering to a subject in need of treatment the pharmaceuticalcomposition of claim 4 thereby preventing or treating the subject'sdisease or disorder.
 13. The method of claim 12, wherein the disease ordisorder is cancer.
 14. The method of claim 13, wherein the cancercomprises an adenocarcinoma of the breast, prostate, and colon;melanoma; non-small cell lung; glioma; leukemia; or cancer of the bone,breast, digestive system, colorectal, liver, pancreatic, pituitary,testicular, orbital, head and neck, central nervous system, acoustic,pelvic, respiratory tract, or urogenital tract.
 15. The method of claim12, wherein the pharmaceutical composition further comprises a cancertherapeutic agent.
 16. The method of claim 15, wherein the cancertherapeutic agent comprises a chemotherapeutic agent or radiotherapeuticagent.
 17. The method of claim 12, wherein the disease or disordercomprises Alzheimer's disease, Parkinson's disease, Huntington'sdisease, stroke, osteoporosis, diabetes or age-related maculardegeneration.